CN111621543A - Method for detecting florfenicol residue - Google Patents
Method for detecting florfenicol residue Download PDFInfo
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- CN111621543A CN111621543A CN202010557535.4A CN202010557535A CN111621543A CN 111621543 A CN111621543 A CN 111621543A CN 202010557535 A CN202010557535 A CN 202010557535A CN 111621543 A CN111621543 A CN 111621543A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
Abstract
The invention discloses a florfenicol residue detection method, and belongs to the technical field of toxicology detection. The method comprises the following steps: and (3) performing an exposure contamination test by using the extracting solution of the sample to be detected to act on the zebra fish embryo, and judging whether the florfenicol in the sample to be detected exceeds the standard or not according to the tissue pathological change condition of the zebra fish embryo. The tissue pathology comprises at least one of a liver pathology, an intestinal pathology, a cardiovascular pathology, and an ocular pathology. The sample to be tested comprises meat products and/or egg products. The method can effectively reduce the cost of the florfenicol residue detection test, improve the simplicity of operation and reduce the environmental risk.
Description
Technical Field
The invention relates to the technical field of toxicological detection, and particularly relates to a method for detecting florfenicol residues.
Background
Florfenicol is a derivative of thiamphenicol, is an artificially synthesized high-efficiency broad-spectrum antibiotic special for animals, has the functions of blood toxicity, embryo toxicity and immunosuppression, and is commonly used for preventing and treating various bacterial diseases of animals. However, due to the irregular use and abuse of the breeding industry, it produces residues in animal food and thus poses a great risk to food safety. In recent years, reports of the standard exceeding of florfenicol detected in pork, poultry, eggs, products thereof and the like are on the rise, so that the problem of residual detection of the florfenicol is also highly concerned by experts at home and abroad.
At present, the method for detecting florfenicol residues generally has high cost and complicated operation.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for detecting florfenicol residues, which can effectively reduce the cost of florfenicol residue detection tests, improve the simplicity of operation and reduce environmental risks.
The invention is realized by the following steps:
in a first aspect, the present application provides a method for detecting florfenicol residues, comprising the following steps:
performing an exposure contamination test by using the extract of the sample to be tested to act on the zebra fish embryo, and judging whether the florfenicol in the sample to be tested exceeds the standard or not according to the tissue lesion condition of the zebra fish embryo;
the tissue pathology comprises at least one of a liver pathology, an intestinal pathology, a cardiovascular pathology, and an ocular pathology.
The sample to be tested comprises meat products and/or egg products.
In an optional embodiment, whether florfenicol in a sample to be detected exceeds the standard or not is judged according to the liver lesion condition of the zebra fish embryo.
Liver disorders include liver loss, liver degeneration and liver cirrhosis.
Taking 10-30 zebra fish embryos as detection samples, and judging whether the standard exceeds the standard or not comprises the following steps:
the incidence rate of liver diseases is 0-30%, which means that florfenicol does not exceed the standard;
the incidence of liver diseases is 30-60%, which indicates that florfenicol exceeds the standard;
the incidence rate of liver diseases is more than 60 percent, which indicates that the florfenicol is seriously out of standard.
The incidence of liver lesion is the number of zebrafish embryos with liver lesion/total number of zebrafish embryos multiplied by 100%.
In an alternative embodiment, the intestinal lesions include missing folds in the intestine, abnormal intestinal size and abnormal color in the lumen of the intestine.
In alternative embodiments, the cardiovascular pathology comprises pericardial edema, abnormal blood flow velocity, or loss of blood circulation.
In an alternative embodiment, the ocular pathology comprises a reduction in ocular pigment.
In an alternative embodiment, the method for preparing the extract of the sample to be tested comprises:
extracting a sample of a sample to be detected by using an extractant, carrying out solid-liquid separation for the first time, carrying out degreasing on a supernatant, carrying out solid-liquid separation for the second time, removing a reagent used for degreasing, evaporating an extractant layer to dryness, and redissolving by using DMSO.
In an alternative embodiment, the supernatant after the first centrifugation is extracted repeatedly with a new extractant, centrifuged again under the conditions of the first centrifugation, and the supernatants are combined for defatting.
In alternative embodiments, the extractant comprises acetonitrile acetate or acetonitrile.
In an alternative embodiment, the mass ratio of extractant to sample is 1-2:100, preferably 1: 100.
In an alternative embodiment, the mass of the sample is 2-5 g.
In an alternative embodiment, the ratio of n-hexane to sample is 2-5g to 20mL, preferably 2 g:20 mL.
In an alternative embodiment, the ratio of DMSO to sample is 2-5g to 200. mu.L, preferably 2 g:200 μ L.
In an alternative embodiment, both the first centrifugation and the second centrifugation are centrifugation, preferably vortex centrifugation.
In an alternative embodiment, the first vortex centrifugation is performed at 10000-.
In an alternative embodiment, the second vortex centrifugation is performed at 10000-.
In an alternative embodiment, fertilized zebrafish eggs are cultured in embryo culture medium and normally developing zebrafish embryos at the same developmental stage are selected for exposure contamination testing.
The selected well-developed zebrafish embryos were non-dead, fertilized and non-malformed.
In alternative embodiments, the embryo culture fluid is in accordance with: pH: 6.0-9.5, salinity: 0.25% -0.50%, conductivity: 200-.
In alternative embodiments, the embryo culture fluid is in accordance with: pH: 7.0-8.0, salinity: 0.25% -0.50%, conductivity: 500-.
In an alternative embodiment, the fertilized egg is obtained by the following steps: naturally mating the female zebra fish adult fish and the male zebra fish adult fish which are sexually mature to obtain fertilized eggs.
In an alternative embodiment, the ratio of adult female zebrafish to adult male zebrafish for natural mating is 1-2: 1-2, preferably 1: 1.
In an alternative embodiment, the day and night rhythm of adult female and adult male zebrafish is controlled: separating adult female zebra fish and adult male zebra fish 20-21 points a day before egg collection in 14 hours in the daytime and 10 hours at night, naturally mating the adult female zebra fish and the adult male zebra fish 8-9 points a morning and collecting fertilized eggs.
Preferably, the day and night rhythm of adult female zebrafish and adult male zebrafish is controlled: the adult female zebra fish and the adult male zebra fish are naturally mated and fertilized eggs are collected at 9 am on the next day after the adult female zebra fish and the adult male zebra fish are separated by 21 points on the day before egg collection in 14 hours in the daytime and 10 hours at night.
In an alternative embodiment, the collected fertilized eggs are washed with embryo culture solution and then placed in the embryo culture solution for constant temperature light-controlled culture at 25-28 ℃.
In an alternative embodiment, the washed fertilized eggs are placed in a culture well plate, 10-30 eggs per well, 10-30mL of embryo culture solution per well, and then incubated under constant temperature and light control.
Preferably, the washed fertilized eggs are placed in a culture well plate, 10 eggs are placed in each well, 10mL of embryo culture solution is added in each well, and then the incubation and light control are carried out.
In an alternative embodiment, the temperature during the cultivation is 28 ℃.
In an alternative embodiment, the intensity of illumination during the culturing is between 54 and 324 lux.
In alternative embodiments, the culture well plate comprises a 6-well plate, a 12-well plate, or a 96-well plate.
In an alternative embodiment, when 2-4hpf, developing normal zebrafish embryos are selected under a microscope; preferably, when 3hpf, developing normal zebrafish embryos are picked under a microscope.
In alternative embodiments, the exposure contamination test comprises: adding 10-30 mL/hole of embryo culture solution, 1-3 muL/hole of venom and 10-30 zebra fish embryos into the to-be-detected hole of the new culture hole plate, and then continuously culturing for 96-120 h; in the culture process, observing and recording the death condition of the zebra fish and the tissue pathological change condition of the zebra fish embryo every day, and removing the dead fish; after the culture is finished, fixing the fry by methylcellulose gel, and observing and counting the death condition and the toxicity end point of the zebra fish.
In an alternative embodiment, the toxicant solution comprises a test solution of unknown toxicity.
Preferably, the contamination solution comprises 0.1% DMSO, matrix-free extract, florfenicol standard solutions with different concentrations, sample extract without adding florfenicol standard solution, and florfenicol matrix matching standard solutions with different concentrations.
In an alternative embodiment, 10 mL/well of embryo culture solution, 1 μ L/well of venom, and 10/well of zebrafish embryos are added to the wells of the culture well plate to be tested.
In an alternative embodiment, the duration of the incubation is 96 h.
In an alternative embodiment, the tissue pathology of the zebrafish embryo is determined every 12-24h during the continuous culture period, preferably every 24 h.
In an alternative embodiment, the method of preparing a methylcellulose gel comprises: stirring methylcellulose and water under heating for 4-8 hr, and cooling.
In alternative embodiments, the ratio of methylcellulose to water may be from 1 to 2 g: 100-200mL, preferably 2 g: 100 mL.
In an alternative embodiment, the heating temperature is 70-90 deg.C, preferably, the heating temperature is 80 deg.C.
In an alternative embodiment, images of liver tissue lesions after zebrafish embryo exposure contamination test were taken by photographic microscopy.
The invention has the following beneficial effects:
the zebra fish is used as a model organism, the extracting solution of a sample to be detected is directly used as a target object of zebra fish exposure detection, the dosage of the zebra fish exposure detection reagent conforms to that of a human body, and reasonable technical support is provided for extrapolation of animal experiment results to the human body.
Whether the florfenicol in the sample to be detected exceeds the standard or not is judged according to the tissue pathological change condition of the zebra fish embryo after the exposure contamination test, particularly the liver pathological change condition, so that a relatively accurate detection result can be obtained, the cost of the florfenicol residue detection test is effectively reduced, the simplicity of operation is improved, and the environmental risk is reduced.
The method can be used as one of the means for detecting florfenicol in a sample to be detected and classifying the safety grade, and provides a new idea and an implementation scheme for veterinary drug residue detection.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a graph showing the results of hepatotoxic effects of zebra fish fries after florfenicol exposure in example 1, wherein A is liver deficiency, B is liver degeneration, C is liver reduction, and D is normal;
FIGS. 2 and 3 show the percentage of abnormal liver disease in zebrafish of each treatment group in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The method for detecting florfenicol residues provided by the present application is specifically described below.
The application provides a florfenicol residue detection method, which comprises the following steps:
and (3) performing an exposure contamination test by using the extracting solution of the sample to be detected to act on the zebra fish embryo, and judging whether the florfenicol in the sample to be detected exceeds the standard or not according to the tissue pathological change condition of the zebra fish embryo.
Wherein the tissue lesion condition comprises at least one of liver lesion, intestinal lesion, cardiovascular lesion and eye lesion.
The sample to be tested comprises meat products and/or egg products, wherein the meat products can comprise pork or poultry (such as chicken).
In an alternative embodiment, whether florfenicol in the sample to be tested exceeds the standard is preferably judged according to the liver lesion condition of the zebra fish embryo.
For reference, the index of liver lesion may include, for example, liver loss, liver degeneration, and liver size reduction.
Correspondingly, 10-30 zebra fish embryos are used as detection samples, and the judgment standard for judging whether the florfenicol is overproof or not comprises the following steps:
when the incidence rate of liver diseases is 0-30%, the florfenicol is not overproof;
when the incidence rate of liver diseases is 30-60%, the florfenicol is out of limits;
when the incidence rate of liver diseases is more than 60 percent, the florfenicol is seriously out of standard.
Wherein, the incidence rate of liver lesion is the number of zebrafish embryos with liver lesion/total number of zebrafish embryos multiplied by 100%.
In addition, in an optional embodiment, when whether florfenicol in a sample to be tested exceeds the standard is judged according to the intestinal lesion condition of the zebra fish embryo, the indicators of the intestinal lesion may include, for example, absence of intestinal folds, abnormal intestinal size, abnormal color of intestinal lumen, and the like.
In an alternative embodiment, when whether florfenicol in a sample to be tested exceeds the standard is judged according to the cardiovascular disease condition of zebra fish embryos, the index of the cardiovascular disease can comprise pericardial edema, abnormal blood flow speed or blood circulation loss and the like.
In an alternative embodiment, when whether florfenicol in a sample to be tested exceeds the standard is judged according to the eye pathological changes of zebra fish embryos, the indexes of the eye pathological changes can include, for example, reduction of eye pigment and the like.
It is worth to be noted that, in the tissue lesion condition judgment types provided by the present application, the hepatotoxicity index sensitivity is the highest, and the hepatotoxicity result and florfenicol present a certain dose effect relationship, so that when detecting whether florfenicol residue exceeds the standard, it is preferable to judge whether florfenicol in the sample to be detected exceeds the standard according to the tissue lesion condition of zebra fish embryos.
In the present application, the method for preparing the extract liquid of the sample to be tested may include, for example:
extracting a sample of a sample to be tested with an extractant, performing first solid-liquid separation (such as first centrifugation), defatting the supernatant, performing second solid-liquid separation (such as second centrifugation), removing the reagent used for defatting, evaporating the extractant layer, and redissolving with DMSO.
In an alternative embodiment, the supernatant from the first centrifugation is extracted repeatedly with a new extractant, centrifuged again under the conditions of the first centrifugation, and the supernatant from this centrifugation is combined with the supernatant from the first centrifugation for defatting.
The extractant may comprise, for example, acetonitrile acetate or acetonitrile.
In alternative embodiments, the mass ratio of extractant to sample may be 1-2:100, preferably 1: 100.
In alternative embodiments, the mass of the sample may be 2-5g, such as 2g, 2.5g, 3g, 3.5g, 4g, 4.5g, or 5g, etc.
In alternative embodiments, the ratio of n-hexane to sample may be 2-5g to 20mL, such as 2 g:20mL, 3 g:20mL, 4 g:20mL or 5g:20mL, etc., preferably 2 g:20 mL.
In alternative embodiments, the ratio of DMSO to sample is 2-5g to 200 μ L, such as 2 g: 200. mu.L, 3 g: 200. mu.L, 4 g: 200. mu.L or 5g: 200. mu.L, etc., preferably 2 g:200 μ L.
In an alternative embodiment, both the first and second centrifugation may be vortex centrifugation.
Wherein the first vortex centrifugation can be performed under the conditions of 10000-12000r/m for 5-8 min. The second vortex centrifugation can be performed at 10000-12000r/m for 1-2 min.
In some embodiments, the extraction may be performed as follows: accurately weighing 2.0-5.0g of homogenized poultry meat sample in a centrifuge tube, adding 1 wt% of acetic acid acetonitrile, performing first vortex centrifugation, and transferring the supernatant into another centrifuge tube. Extracting repeatedly, centrifuging, mixing the supernatants, adding n-hexane for defatting, vortex centrifuging, removing n-hexane, transferring acetonitrile layer into heart bottle, rotary evaporating to dryness, and dissolving in DMSO.
In an alternative embodiment, fertilized zebrafish eggs are cultured in embryo culture medium and normally developing zebrafish embryos at the same developmental stage are selected for exposure contamination testing.
The selected well-developed zebrafish embryos were non-dead, fertilized and non-malformed.
In alternative embodiments, the embryo culture fluid is, for example, in accordance with: pH: 6.0-9.5, salinity: 0.25% -0.50%, conductivity: 200-.
Preferably, the embryo culture fluid meets the following conditions: pH: 7.0-8.0, salinity: 0.25% -0.50%, conductivity: 500-.
In an alternative embodiment, the fertilized egg is obtained by the following steps: naturally mating the female zebra fish adult fish and the male zebra fish adult fish which are sexually mature to obtain fertilized eggs.
By reference, the ratio of adult female zebrafish to adult male zebrafish for natural mating may be 1-2: 1-2, preferably 1: 1.
In an alternative embodiment, the day and night rhythm of adult female and adult male zebrafish is controlled: separating adult female zebra fish and adult male zebra fish 20-21 points a day before egg collection in 14 hours in the daytime and 10 hours at night, naturally mating the adult female zebra fish and the adult male zebra fish 8-9 points a morning and collecting fertilized eggs.
Preferably, the day and night rhythm of adult female zebrafish and adult male zebrafish is controlled: the adult female zebra fish and the adult male zebra fish are naturally mated and fertilized eggs are collected at 9 am on the next day after the adult female zebra fish and the adult male zebra fish are separated by 21 points on the day before egg collection in 14 hours in the daytime and 10 hours at night.
In an alternative embodiment, the collected fertilized eggs are washed with embryo culture solution and then placed in the embryo culture solution for constant temperature light-controlled culture at 25-28 ℃.
In an alternative embodiment, the washed fertilized eggs are placed in a culture well plate, 10-30 eggs per well, 10-30mL of embryo culture solution per well, and then incubated under constant temperature and light control.
In order to improve the detection accuracy, the cleaned fertilized eggs can be placed in a culture well plate, 10 fertilized eggs are placed in each well, 10mL of embryo culture solution is added in each well, and then the fertilized eggs are cultured under the condition of constant temperature and light control.
Preferably, the temperature during the culture can be thermostatically controlled to 28 ℃.
In an alternative embodiment, the light conditions during the culture may be, for example, a light intensity of 54 to 324lux, preferably a light intensity of 200 lux.
In alternative embodiments, a 6-well plate, a 12-well plate, or a 96-well plate may be used as the culture well plate, and a 6-well plate is preferably used to improve the accuracy of the detection result.
In an alternative embodiment, when 2-4hpf, developing normal zebrafish embryos are selected under a microscope; preferably, when 3hpf, developing normal zebrafish embryos are picked under a microscope.
In alternative embodiments, exposure contamination tests may include, for example: adding 10-30 mL/well of embryo culture solution, 1-3 μ L/well of venom and 10-30 zebra fish embryos into the wells to be tested of the new culture well plate, and then continuously culturing for 96-120 h. During the culture process, the death condition of the zebra fish and the tissue pathological change condition of the zebra fish embryo are observed and recorded every day, and the dead fish are removed. After the culture is finished, fixing the fry by methylcellulose gel, and observing and counting the death condition and the toxicity end point of the zebra fish.
In an alternative embodiment, the contamination solution may comprise, for example, a test solution of unknown toxicity.
Preferably, the contamination solution comprises 0.1% DMSO, matrix-free extract, florfenicol standard solutions with different concentrations, sample extract without adding florfenicol standard solution, and florfenicol matrix matching standard solutions with different concentrations.
In order to improve the detection accuracy, 10 mL/hole of embryo culture solution, 3 mu L/hole of venom and 10/hole of zebra fish embryos are added into the to-be-detected holes of the culture hole plate.
In an alternative embodiment, the duration of the incubation is 96 h.
In an alternative embodiment, the tissue pathology of the zebrafish embryo is determined every 12-24h during the continuous culture period, preferably every 24 h.
As a reference, the above-mentioned method for preparing a methylcellulose gel may, for example, comprise: stirring methylcellulose and water under heating for 4-8 hr, and cooling.
Wherein, the dosage ratio of the methyl cellulose to the water can be 1-2 g: 100-200mL, preferably 2 g: 100 mL.
The heating temperature may be 70 to 90 deg.C, preferably, the heating temperature is 80 deg.C.
The methylcellulose gel prepared by the method is clear and transparent jelly.
In an alternative embodiment, images of liver tissue lesions after zebrafish embryo exposure contamination test were taken by photographic microscopy.
In the application, zebra fish is taken as a model organism, the extract of the sample to be detected is subjected to an exposure contamination test on zebra fish embryos, whether florfenicol in the sample to be detected exceeds the standard or not is judged according to the tissue pathological change condition of the zebra fish embryos, particularly the liver pathological change condition, and a relatively accurate detection result can be obtained (wherein the liver toxicity index sensitivity is high, the sample to be detected generates relatively obvious liver toxicity on the zebra fish, and the dose effect relationship exists with the florfenicol concentration), so that the florfenicol residue detection test cost is effectively reduced, the simplicity of operation is improved, and the environmental risk is reduced.
It is worth to be noted that, in the process of evaluating the florfenicol residual degree in the sample to be detected, the florfenicol matrix matching standard solution and the florfenicol standard solution can be simultaneously arranged to replace the embryo culture solution, and the zebra fish embryo is cultured according to the same steps and conditions to compare the detection result.
Specifically, the florfenicol matrix matching standard solution is a solution obtained by adding a florfenicol standard solution into an extracting solution of a sample to be detected. The preparation method can specifically adopt the following steps: adding a certain amount of florfenicol standard solution with the concentration of 1-500mg/L into the extracting solution of a sample to be detected respectively, carrying out rotary evaporation until the solution is dry, and re-dissolving the solution by DMSO. The preparation of florfenicol standard solutions can then be referred to: and additionally, carrying out liquid nitrogen blowing on the florfenicol standard solution with the same concentration gradient in the preparation process of the florfenicol matrix matching standard solution until the florfenicol standard solution is dried, and re-dissolving the florfenicol standard solution by using DMSO.
Bearing, compare with prior art, the advantage of this application includes:
(1) the application directly uses the extract of the poultry product as the target object of zebra fish exposure detection, and the dosage of the zebra fish exposure detection is consistent with that of the zebra fish contact, thereby providing reasonable technical support for animal experiment results to be extrapolated to human beings.
(2) The method can reduce the cost of florfenicol residue detection in poultry products, is simple and convenient to operate, and has small environmental risk. The application has a good agent-effect relationship, and the zebra fish can be used as one of means for detecting the poultry product florfenicol and classifying the safety level, so that a new thought and an implementation scheme are provided for veterinary drug residue detection.
According to the limit standard of veterinary drug residues in China and the European Union, the limit of florfenicol (and florfenicol amine in total) in the poultry meat is 0.1 mg/kg. When the concentration of the florfenicol standard solution and the concentration of the florfenicol matrix matching standard solution are 0.1mg/L, the incidence rate of the toxicity of the zebra fish liver is 50 percent and 60 percent respectively. The florfenicol toxicity judgment standard in chicken can be as follows: the incidence rate of hepatotoxicity is 0-30%, which means that the florfenicol is not out of standard; the incidence rate of hepatotoxicity is 30-60% of the standard exceeding; the incidence of hepatotoxicity > 60% is severely exceeded.
The features and properties of the present invention are described in further detail below with reference to examples. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Method for evaluating florfenicol residual degree in chicken by adopting zebra fish hepatotoxicity index
A1: obtaining the chicken sample extract. Accurately weighing 2.0g of the homogenized chicken sample into a 50mL centrifuge tube, adding 10mL of 1 wt% acetic acid acetonitrile, performing vortex extraction for 2min, centrifuging at 10000r/m for 5min (the temperature is set as 4 ℃), and transferring the supernatant into another 50mL centrifuge tube. Extracting once again, centrifuging, mixing the supernatants, adding 20mL of n-hexane, vortex mixing for 1min, centrifuging at 10000r/m for 1min (temperature set at 4 ℃), removing n-hexane, transferring the acetonitrile layer into a chicken heart bottle, rotary steaming in water bath at 40 ℃ until dry, and re-dissolving with 200 μ L of DMSO.
A2: obtaining the florfenicol matrix matching standard solution. Accurately weighing 2.0g of the homogenized chicken sample into a 50mL centrifuge tube, adding 10mL of 1 wt% acetic acid acetonitrile, performing vortex extraction for 2min, centrifuging at 10000r/m for 5min (the temperature is set as 4 ℃), and transferring the supernatant into another 50mL centrifuge tube. The extraction is repeated once, the supernatant is merged after centrifugation, 20mL of normal hexane is added, vortex mixing is carried out for 1min, centrifugation is carried out at 10000r/m for 1min (the temperature is set as 4 ℃), the normal hexane is removed, the acetonitrile layer is transferred into a heart bottle, and 200 mu L of florfenicol standard solutions of 5mg/L, 20mg/L, 50mg/L, 100mg/L and 200mg/L are respectively added into the heart bottle. Spin-evaporate to dryness in a water bath at 40 ℃ and redissolve with 200. mu.L DMSO.
B: collecting zebra fish embryos. Controlling day and night rhythm of adult zebra fish, wherein the day is 14 hours, the night is 10 hours, and the day before egg collection is 21: 00 sex-matured zebra fish is cultured according to the male-female ratio of 1:1, transferring the fertilized eggs into a spawning vat at a ratio of 9:00 a.m. the next day, naturally mating and collecting the fertilized eggs, cleaning the fertilized eggs collected twice by using embryo culture solution to remove impurities, selecting zebra fish embryos which normally develop and have consistent development period under a microscope when the height of the fertilized eggs is 3hpf, placing the zebra fish embryos in the embryo culture solution, and carrying out light-control culture at 28 ℃.
Wherein, the embryo culture solution meets the following conditions: pH: 7.5, salinity: 0.25%, conductivity: 600. mu.S/cm.
The light intensity during the culture was 200 lux.
C: and (5) carrying out a zebra fish embryo exposure contamination test. And D, placing the zebra fish embryos collected in the step B into a 6-well plate, wherein 10 embryos are added into each well, and 10mL of embryo culture water is added. The experiment was set up with a normal control group (CK) (embryo culture water-treated group), a solvent control group (3. mu.L of 0.1% DMSO was added), a process blank group (SCK) (3. mu.L of matrix-free extract was added), a florfenicol standard solution group (3. mu.L of florfenicol standard solutions of different concentrations were added, 5mg/L, 20mg/L, 50mg/L, 100mg/L and 200mg/L, respectively), a matrix blank group (MCK) (3. mu.L of sample extract without florfenicol standard solution added), a matrix spiked group (MTD) (3. mu.L of florfenicol matrix-matched standard solutions of different concentrations, 5mg/L, 20mg/L, 50mg/L, 100mg/L and 200mg/L, respectively, 5 concentrations), three of each treatment set up in parallel and were incubated in a 28 ℃ incubator. In the experimental process, observing and recording the death condition of the zebra fish every 24h, removing the dead fish, and taking and recording abnormal conditions; after exposure for 96h, the larvae are fixed by methylcellulose gel, the death condition and hepatotoxicity effect of the zebra fish are observed and counted, and the photos are taken for recording.
The composition of the virus staining solution is 0.1% DMSO, matrix-free extracting solution, florfenicol standard solutions with different concentrations, sample extracting solution without adding florfenicol standard solution, and florfenicol matrix matching standard solutions with different concentrations.
The methylcellulose gel is obtained by the following steps: mixing methyl cellulose and water in a ratio of 2 g: mixing and stirring at 80 deg.C for 4 hr at a ratio of 100mL, and cooling.
By analyzing the results of example 1, it was found that florfenicol and chicken samples with florfenicol added had the following effects on zebrafish embryos in this example:
liver abnormalities include liver loss, liver diminution, and liver degeneration, as shown in figure 1. The statistical results of the liver abnormality rates of the respective groups are shown in table 1, and the line graphs are plotted based on the statistical results as shown in fig. 2 and 3.
TABLE 1 statistical results of liver abnormality rates
TABLE 1 statistical results of (continuous) liver abnormality rates
As can be seen from fig. 2 and fig. 3 (the upper and lower straight dashed lines in the figure are respectively the severe superscalar and superscalar boundary, and the broken line is the incidence of liver abnormality of different treatment groups): after exposure for 96 hours, the liver abnormality rates of the chicken sample extract exposure groups containing 20 mug/L, 50 mug/L, 100 mug/L and 200 mug/L florfenicol are all above 50%, and the liver abnormality rates of other groups are in the range of 0-30%. In the florfenicol standard solution exposure groups with different concentrations, the liver abnormality rate of the zebra fish is more than 50% only when the concentration reaches 100 mug/L and 200 mug/L. Therefore, the liver toxicity is increased to a certain extent by adding the chicken matrix extracting solution, and when the florfenicol content in the chicken reaches the limit of 100 mug/L of veterinary drug residue in China and European Union, the liver abnormality rate of the zebra fish is 60%, and 50% of liver abnormality of the zebra fish can be caused by 100 mug/L of florfenicol standard solution.
Example 2: selection of Zebra Fish toxicity index to assess florfenicol residual levels in Chicken
The procedure is as in example 1.
By analyzing the results of example 2, it was found that florfenicol and chicken samples with florfenicol added had the following effects on zebrafish embryos in this example:
after the florfenicol standard solution is exposed to zebrafish embryos for 96 hours, hepatotoxicity such as liver deletion, liver reduction and liver degeneration, enterotoxicity such as intestinal tract wrinkle deletion, intestinal tract size abnormality and intestinal tract lumen color abnormality, cardiovascular toxicity such as pericardial edema, blood flow slowness and blood circulation deletion and abnormality such as eye pigment reduction are observed. Wherein, the incidence rate of hepatotoxicity presents a certain dose effect relationship, and the LC50 is 194.5 mug/L.
After the florfenicol matrix matching standard solution is exposed to zebra fish embryos for 96 hours, hepatotoxicity such as liver deletion, liver reduction and liver degeneration, enterotoxicity such as abnormal intestinal tract size and abnormal color of intestinal tract lumen, developmental toxicity such as shortened body length and bent trunk, cardiovascular toxicity such as slow blood flow and blood circulation deletion, reduction of eye pigment, embryonic death and the like are observed. Too fast a speed of the zebrafish embryo exfoliation was observed at 48h exposure. Wherein, the incidence rate of hepatotoxicity, the incidence rate of ocular abnormality and the mortality rate present a certain dose-effect relationship, and LC50 is respectively 11.2 mug/L, 10.2 mug/L and 11.0 mug/L.
The result shows that the hepatotoxicity index has high sensitivity, can be observed in two groups of exposure experiments, and presents a certain dose effect relationship, so the hepatotoxicity of the zebra fish embryo is preferably used as the toxicity index for evaluating the florfenicol residual degree in chicken.
In conclusion, the zebra fish is taken as a model organism and the extracting solution of the sample to be detected is directly taken as a target object for zebra fish exposure detection, the dosage of the zebra fish exposure detection reagent is consistent with that of the zebra fish, and reasonable technical support is provided for extrapolation of animal experiment results to human. Whether the florfenicol in the sample to be detected exceeds the standard or not is judged according to the tissue pathological change condition of the zebra fish embryo after the exposure contamination test, particularly the liver pathological change condition, so that a relatively accurate detection result can be obtained, the cost of the florfenicol residue detection test is effectively reduced, the simplicity of operation is improved, and the environmental risk is reduced. The method can be used as one of the means for detecting florfenicol in a sample to be detected and classifying the safety grade, and provides a new idea and an implementation scheme for veterinary drug residue detection.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A florfenicol residue detection method is characterized by comprising the following steps:
performing an exposure contamination test on the extract of a sample to be tested by acting on the zebra fish embryo, and judging whether the florfenicol in the sample to be tested exceeds the standard or not according to the tissue lesion condition of the zebra fish embryo;
the tissue lesion condition comprises at least one of a liver lesion, an intestinal lesion, a cardiovascular lesion and an eye lesion;
preferably, the sample to be tested comprises a meat product and/or an egg product;
more preferably, the sample to be tested comprises chicken.
2. The detection method according to claim 1, wherein whether florfenicol in the sample to be detected is overproof or not is judged according to liver lesion conditions of the zebra fish embryos;
the liver pathological changes comprise liver loss, liver degeneration and liver reduction;
taking 10-30 zebra fish embryos as detection samples, and judging whether the standard exceeds the standard or not comprises the following steps:
the incidence rate of liver diseases is 0-30%, which means that florfenicol does not exceed the standard;
the incidence of liver diseases is 30-60%, which indicates that florfenicol exceeds the standard;
the incidence rate of liver diseases is more than 60 percent, which indicates that the florfenicol seriously exceeds the standard;
the incidence rate of liver lesion is the number of zebrafish embryos with liver lesion/total number of zebrafish embryos multiplied by 100%.
3. The method according to claim 1 or 2, wherein the preparation method of the extract solution of the sample to be tested comprises:
extracting a sample of the sample to be detected by using an extractant, carrying out solid-liquid separation for the first time, carrying out degreasing on a supernatant, carrying out solid-liquid separation for the second time, removing a reagent used for degreasing, evaporating an extractant layer to dryness, and redissolving by using DMSO (dimethyl sulfoxide);
preferably, the supernatant after the first centrifugation is repeatedly extracted by using a new extracting agent, the supernatant is centrifuged again according to the condition of the first centrifugation, and the supernatants are combined for degreasing;
preferably, the extractant comprises acetonitrile acetate or acetonitrile;
preferably, the mass ratio of the extractant to the sample is 1-2:100, more preferably 1: 100;
preferably, the mass of the sample is 2-5 g;
preferably, the reagents used for degreasing include n-hexane;
preferably, the ratio of the amount of the n-hexane to the amount of the sample is 2-5g to 20mL, more preferably 2 g:20 mL;
preferably, the ratio of the amount of DMSO to the amount of the sample is 2-5g: 200. mu.L, more preferably 2 g:200 mu L;
preferably, the first solid-liquid separation and the second solid-liquid separation are both centrifugal separation, and more preferably, vortex centrifugation;
preferably, the first vortex centrifugation is performed under the conditions of 10000-;
preferably, the second vortex centrifugation is performed at 10000-.
4. The detection method according to claim 1 or 2, wherein fertilized eggs of zebra fish are cultured in an embryo culture solution, and normally-developed zebra fish embryos at the same developmental stage are selected for the exposure contamination test;
the normal-developing zebrafish embryo is an undecided, fertilized and malformed embryo.
5. The assay of claim 4, wherein the exposure contamination test comprises: adding 10-30 mL/hole of embryo culture solution, 1-3 muL/hole of venom and 10-30 zebra fish embryos into the to-be-detected holes of the culture well plate, and then continuously culturing for 96-120 h; in the culture process, observing and recording the death condition of the zebra fish and the tissue pathological change condition of the zebra fish embryo every day, and removing the dead fish; after the culture is finished, fixing the fry by methylcellulose gel, and observing and counting the death condition and the toxicity end point of the zebra fish.
6. The assay method according to claim 5, wherein 10 mL/well of the embryo culture solution, 1 μ L/well of the venom solution, and 10/well of the zebrafish embryos are added to the wells to be assayed of the culture well plate.
7. The assay of claim 5, wherein the incubation time is 96 hours.
8. The detection method as claimed in claim 5, wherein the tissue lesion of the zebrafish embryo is determined every 12-24h during the continuous culture period;
preferably, the histological condition of the zebrafish embryos is determined every 24 h.
9. The detection method according to claim 5, wherein the preparation method of the methylcellulose gel comprises: stirring methylcellulose and water under heating for 4-8h, and cooling;
preferably, the dosage ratio of the methyl cellulose to the water is 1-2 g: 100-200mL, more preferably 2 g: 100 mL;
preferably, the heating temperature is 70-90 deg.C, more preferably, the heating temperature is 80 deg.C.
10. The method of claim 5, wherein the pathological condition of liver tissue after the zebrafish embryo exposure contamination test is imaged by a photographic microscope.
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