CN112798778A - Immunofluorescence chromatography detection card and method for synchronously detecting florfenicol and trimethoprim contents in poultry eggs - Google Patents
Immunofluorescence chromatography detection card and method for synchronously detecting florfenicol and trimethoprim contents in poultry eggs Download PDFInfo
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Abstract
The invention discloses an immunofluorescence chromatography detection card and a method for synchronously detecting the content of florfenicol and trimethoprim in poultry eggs; the immunofluorescence chromatography detection card and the immunofluorescence chromatography detection method are simple and time-saving in sample pretreatment operation, free of blow-drying, high in detection method sensitivity, suitable for on-site instant detection, and capable of synchronously detecting florfenicol and trimethoprim contents in poultry eggs, and technically characterized by comprising a bottom plate, wherein a water absorption paper pad, a reaction membrane, a combination pad and a sample pad are sequentially connected to the bottom plate from left to right, the reaction membrane is marked with three lines of a florfenicol detection line T1, a trimethoprim detection line T2 and a quality control C line, and the combination pad is coated with florfenicol detection microspheres, trimethoprim detection microspheres and goat anti-chicken IgY polyclonal antibody labeled microspheres; belongs to the technical field of biological detection.
Description
Technical Field
The invention discloses an egg detection method, in particular to an immunofluorescence chromatography detection card and method for synchronously detecting the content of florfenicol and trimethoprim in eggs, belonging to the technical field of biological detection.
Background
Florfenicol is a novel broad-spectrum antibiotic of chloramphenicol, has obvious advantages in safety and effectiveness, and is widely used in livestock and poultry breeding industry. Trimethoprim is named trimethoprim, belongs to a broad-spectrum antibacterial synergist, has weak bactericidal capability, has obvious synergistic effect when being used together with sulfonamides and some antibiotics, and is widely used in livestock and poultry breeding industry.
However, the florfenicol and trimethoprim are used in a large amount, so that the detection rate of the florfenicol and trimethoprim in livestock and poultry products is high, and the public health is seriously threatened, so that the national standard of food safety sets a strict detection limit for the two antibiotics, and the egg laying period of the laying hens is forbidden.
At present, in the prior art, florfenicol and trimethoprim detection chromatograms, enzyme-linked methods, immunochromatography and the like are used, some methods depend on high-end equipment seriously, some methods have high requirements on samples to be detected, the pretreatment is complex, or only one of the methods can be detected, so that the working efficiency is reduced seriously.
Disclosure of Invention
In view of the above problems, a first object of the present invention is to provide an immunofluorescent layer assay card which has high assay sensitivity, is easy and time-saving to operate, does not need to be dried, has high sensitivity, is suitable for on-site real-time assay, and can synchronously assay the florfenicol and trimethoprim contents in poultry eggs.
The invention also aims to provide a method for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs, which is simple to operate, can effectively eliminate interference items, improves the detection sensitivity and is suitable for poultry egg detection in any place.
In order to achieve the above object, a first technical solution provided by the present invention is as follows:
an immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in poultry eggs comprises a bottom plate, wherein a water absorption paper pad, a reaction membrane, a combination pad and a sample pad are sequentially connected to the bottom plate from left to right, the reaction membrane is marked with a florfenicol detection line T1, a trimethoprim detection line T2 and a quality control C line, and the combination pad is coated with florfenicol detection microspheres, trimethoprim detection microspheres and goat anti-chicken IgY polyclonal antibody labeled microspheres;
the florfenicol detection microsphere is prepared by combining a long-chain biotinylated florfenicol monoclonal antibody connected with a polyethylene glycol long-chain lengthened arm (-dPEG 24-) and a streptavidin fluorescent microsphere through biotin and streptavidin;
the trimethoprim detection microsphere is prepared by combining a long-chain biotinylated trimethoprim monoclonal antibody connected with a polyethylene glycol long-chain lengthened arm (-dPEG 24-) and a streptavidin fluorescent microsphere through biotin and streptavidin.
Further, the immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs is prepared by the following steps:
(1) diluting chicken IgY to 0.1mg/mL by using 0.01M phosphate buffer solution with 3% sucrose sugar and pH7.4 to serve as quality control C-line working solution;
(2) diluting the florfenicol coupling antigen to 0.2mg/mL by using a phosphate buffer solution of 0.01M with pH7.4 and containing 3% sucrose sugar to be used as a working solution of a florfenicol detection line;
(3) diluting the trimethoprim coupling antigen to 0.2mg/mL by using a phosphate buffer solution of 0.01M and pH7.4 containing 3% sucrose sugar, and using the diluted sample as a working solution of a trimethoprim detection line;
(4) scribing three lines of a florfenicol detection line T1, a trimethoprim detection line T2 and a quality control line C on a nitrocellulose membrane by using a gold spraying membrane scribing instrument, wherein the distance between two adjacent lines is 5mm, and the scribing concentration is 1 mul/cm; after finishing the scraping, the paper is dried in a drying oven at 45 ℃ for 48 hours.
Further, the immunofluorescence chromatography detection card for synchronously detecting the content of the florfenicol and the trimethoprim in the poultry eggs is characterized in that the sample pad is prepared by the following steps:
(1) preparing 0.02M phosphate buffer solution of pH7.4 containing 1% casein, 0.15M NaCl, 1% PEG 8000 and 1.5% Triton X-100 as a treatment solution;
(2) treating fluid prepared in step (1) at a concentration of 50. mu.l/cm2Uniformly coating the mixture on glass fiber RB65, and drying for 16h at 45 ℃.
Further, the immunofluorescence chromatography detection card for synchronously detecting the content of the florfenicol and the trimethoprim in the poultry eggs is prepared by the following steps: mixing florfenicol detection microspheres, trimethoprim detection microspheres and goat anti-chicken IgY polyclonal antibody marking microspheres, uniformly spraying onto a glass fiber membrane 8964 pad with the width of 1.2cm, and drying at 45 ℃.
Further, the immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs is prepared by the following steps: :
(1) adding 1mL of 0.05M MES buffer solution into a 2mL centrifuge tube, adding 1mg of fluorescent microspheres, and uniformly mixing by vortex; adding 4. mu.l NHS (25 mg/ml) and 4. mu.l EDC (25 mg/ml) into the centrifuge tube, mixing uniformly by vortex, and reacting for 20min on a rotary incubator;
(2) centrifugally separating the activated fluorescent microspheres, removing supernatant, taking precipitate, adding 1000 mu l of 0.02M borate buffer solution with pH8.0, performing ultrasonic treatment for 2s, performing interval for 5s, and repeating the step for 5 times to break up the microspheres;
(3) adding 100 μ g goat anti-chicken IgY, mixing uniformly by vortex, placing on a rotary incubator, and reacting for 60 min;
(4) adding 100 μ l of 10% casein blocking solution into each tube, mixing uniformly by vortex, placing on a rotary incubator after completing ultrasonic treatment, and sealing for 60 min; and centrifuging again, sucking supernatant, retaining precipitate, adding 1mL of microsphere preservation solution, and mixing completely for later use.
Further, the preparation process of the immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry egg comprises three steps:
the first step is as follows: the preparation method of the long-chain biotinylation florfenicol monoclonal antibody and the long-chain biotinylation trimethoprim monoclonal antibody comprises the following steps:
(1) taking out long-chain Biotin (NHS-dPEG 24-Biotin), balancing for 30min at room temperature, and dissolving with DMSO to a final concentration of 10mM for use;
(2) respectively taking 1ml of florfenicol monoclonal antibody (2 mg/ml) and 1ml of trimethoprim monoclonal antibody (2 mg/ml) into a 2ml centrifuge tube, adding 27 mu l of Biotin NHS-dPEG24-Biotin prepared in the previous step into each tube, uniformly mixing by vortex, and reacting for 30min on a rotary incubator;
(3) transferring the mixture to an ultrafiltration tube after the reaction is finished, centrifuging the mixture for 15min at 12000g, and discarding waste liquid;
(4) adding 2ml of PBS buffer solution into the ultrafiltration tube, 12000g, centrifuging 15ml, and discarding waste liquid;
(5) repeating the step (4) for 5 times, re-dissolving 200 mu l of PBS, collecting the labeled antibody, and measuring the concentration by using a BCA kit.
The second step is that: the preparation method of the streptavidin fluorescent microsphere comprises the following steps:
(1) adding 1mL of marking buffer solution 0.05M MES buffer solution into a 2mL centrifuge tube, adding 1mg of fluorescent microspheres, and uniformly mixing by vortex; adding 4 μ l NHS (25 mg/ml) and 4 μ l EDC (25 mg/ml), mixing by vortex, and reacting for 20min on a rotary incubator;
(2) centrifugally separating the activated fluorescent microspheres, discarding supernatant, taking precipitate, adding 1000 μ l of borate buffer solution, performing ultrasonic treatment for 2s, performing intermittent treatment for 5s, and repeating the steps for 5 times to break up the microspheres;
(3) adding 100 mu g of streptavidin into the activated microspheres, uniformly mixing by vortex, and placing on a rotary incubator for reaction for 60 min;
(4) adding 100 μ l of 10% casein blocking solution into each tube, mixing uniformly by vortex, placing on a rotary incubator after ultrasonic treatment, sealing for 60min, centrifuging, removing supernatant, and collecting precipitate;
(5) adding 1mL of microsphere preservation solution into the precipitate, fully and uniformly mixing, carrying out centrifugal separation, sucking supernatant, and taking the precipitate;
(6) repeating the step (5) for 2-3 times to fully remove unreacted streptavidin; then adding 1mL of microsphere storage solution, and fully and uniformly mixing for later use.
The third step: the preparation process of the florfenicol detection microsphere and the trimethoprim detection microsphere comprises the following steps:
(1) respectively adding 5 mu g of each long-chain biotinylation florfenicol monoclonal antibody and long-chain biotinylation trimethoprim monoclonal antibody into 2 tubes of fluorescent microspheres containing 1ml of streptavidin, uniformly mixing in a vortex manner, and reacting for 30min on a rotary incubator;
(2) after the reaction is finished, centrifuging at 16500rpm for 20min, sucking the supernatant, and taking the precipitate;
(3) adding 1mL of microsphere preservation solution, mixing well, centrifuging at 16500rpm for 20min, and removing supernatant;
(4) repeating the step (3) for 3 times, and fully removing the unreacted biotin-labeled antibody;
(5) adding 1mL of microsphere storage solution, and mixing well for later use.
The second technical scheme provided by the invention is as follows:
a method for synchronously detecting the content of florfenicol and trimethoprim in poultry eggs comprises the following steps:
firstly), pretreating a sample to obtain a solution to be detected;
secondly), adding a solution to be detected into the detection card of claim 1, horizontally standing for 10min, and then inserting the solution into a fluorescence immunochromatographic instrument to read the result;
and thirdly), the result is brought into the standard curve, and the content of the florfenicol and the trimethoprim in the sample can be calculated.
Wherein, the pretreatment method in the step one) comprises the following steps:
(1) adding the extract into the fully homogenized egg to-be-detected liquid, manually shaking and uniformly mixing for extraction, centrifuging and taking supernatant;
(2) adding n-hexane and a redissolution into the supernatant, shaking, standing and layering to obtain a lower water phase as a solution to be detected;
in the method for synchronously detecting the content of the florfenicol and the trimethoprim in the poultry eggs, the extracting solution is ethyl acetate containing 0.5 to 1.5 weight percent of trichloroacetic acid.
In the method for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs, the complex solution is PBS buffer solution containing 0.15M NaCl and 0.05M PB, and the pH value is 6.6.
According to the method for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs, the volume ratio of the supernatant to the n-hexane to the complex solution is as follows: 1:5:0.5-1.
Compared with the prior art, the technical scheme provided by the invention has the following technical advantages:
1. the technical scheme provided by the invention is that a long-chain biotinylation monoclonal antibody and a streptavidin fluorescent microsphere are used for preparing a labeled microsphere through the affinity action of Biotin and streptavidin, the long-chain Biotin (NHS-dPEG 24-Biotin) is connected with a polyethylene glycol long-chain lengthened arm (-dPEG 24-), and the other end of the lengthened arm is connected with NHS for coupling and labeling the monoclonal antibody, so that the steric hindrance effect can be effectively reduced, the labeling efficiency is improved, and a Biotin-streptavidin system has the signal amplification effect and can further improve the detection sensitivity.
2. The technology provided by the invention can be used for directly loading the sample for detection after being extracted by a liquid-liquid extraction method, the pretreatment operation is simple, the processes of blow drying, column passing and the like are not needed, the content of the florfenicol and the trimethoprim can be synchronously detected, and the application range is wide.
3. The technology provided by the invention can synchronously detect the content of the florfenicol and the trimethoprim.
Drawings
FIG. 1 is a schematic diagram of a test card structure and test principle provided in the present application;
FIG. 2 is a schematic representation of the principle of the long chain biotinylated monoclonal antibody provided herein;
FIG. 3 is a schematic diagram of the preparation process of streptavidin fluorescent microspheres;
FIG. 4 is a schematic diagram of the preparation process of detection microspheres
Detailed Description
The following claims are hereby incorporated into the detailed description of the invention with further limitations, but not to be construed as limiting the invention in any way, so that any and all modifications that come within the scope of the claims are to be embraced by the invention.
Example 1
The immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in poultry eggs provided by the embodiment comprises a bottom plate, wherein a water absorption paper pad, a reaction membrane, a combination pad and a sample pad are sequentially connected to the bottom plate from left to right, the reaction membrane is marked with three lines of a florfenicol detection line T1, a trimethoprim detection line T2 and a quality control C line, and the combination pad is coated with florfenicol detection microspheres, trimethoprim detection microspheres and goat anti-chicken IgY polyclonal antibody labeled microspheres;
the florfenicol detection microsphere is prepared by combining a long-chain biotinylated florfenicol monoclonal antibody connected with a polyethylene glycol long-chain lengthened arm (-dPEG 24-) and a streptavidin fluorescent microsphere through biotin and streptavidin;
the trimethoprim detection microsphere is prepared by combining a long-chain biotinylated trimethoprim monoclonal antibody connected with a polyethylene glycol long-chain lengthened arm (-dPEG 24-) and a streptavidin fluorescent microsphere through biotin and streptavidin.
Wherein:
the reaction membrane is prepared by the following method:
(1) diluting chicken IgY to 0.1mg/mL by using 0.01M phosphate buffer solution with 3% sucrose sugar and pH7.4 to serve as quality control C-line working solution;
(2) diluting the florfenicol coupling antigen to 0.2mg/mL by using a phosphate buffer solution of 0.01M with pH7.4 and containing 3% sucrose sugar to be used as a working solution of a florfenicol detection line;
(3) diluting the trimethoprim coupling antigen to 0.2mg/mL by using a phosphate buffer solution of 0.01M and pH7.4 containing 3% sucrose sugar, and using the diluted sample as a working solution of a trimethoprim detection line;
(4) scribing three lines of a florfenicol detection line, a trimethoprim detection line and a quality control C line on a nitrocellulose membrane by using a gold spraying membrane scribing instrument, wherein the distance between two adjacent lines is 5mm, and the scribing concentration is 1 mul/cm; after finishing the scraping, the paper is dried in a drying oven at 45 ℃ for 48 hours.
The preparation method of the sample pad comprises the following steps:
(1) preparing 0.02M phosphate buffer solution of pH7.4 containing 1% casein, 0.15M NaCl, 1% PEG 8000 and 1.5% Triton X-100 as a treatment solution;
(2) treating fluid prepared in step (1) at a concentration of 50. mu.l/cm2Uniformly coating the mixture on glass fiber RB65, and drying for 16h at 45 ℃.
The goat anti-chicken IgY polyclonal antibody labeled microsphere is prepared by the following steps:
(1) adding 1mL of 0.05M MES buffer solution into a 2mL centrifuge tube, adding 1mg of fluorescent microspheres, and uniformly mixing by vortex; adding 4. mu.l NHS (25 mg/ml) and 4. mu.l EDC (25 mg/ml) into the centrifuge tube, mixing uniformly by vortex, and reacting for 20min on a rotary incubator;
(3) centrifugally separating the activated fluorescent microspheres, removing supernatant, taking precipitate, adding 1000 mu l of 0.02M borate buffer solution with pH8.0, performing ultrasonic treatment for 2s, performing interval for 5s, and repeating the step for 5 times to break up the microspheres;
(4) adding 100 μ g goat anti-chicken IgY, mixing uniformly by vortex, placing on a rotary incubator, and reacting for 60 min;
(5) adding 100 μ l of 10% casein blocking solution into each tube, mixing uniformly by vortex, placing on a rotary incubator after completing ultrasonic treatment, and sealing for 60 min; centrifuging again, sucking out supernatant, retaining precipitate, adding 1mL of microsphere preservation solution, and mixing well for later use;
the method for preparing the long-chain biotinylated florfenicol monoclonal antibody and the long-chain biotinylated trimethoprim monoclonal antibody comprises the following steps:
(1) taking out long-chain Biotin (NHS-dPEG 24-Biotin), balancing for 30min at room temperature, and dissolving with DMSO to a final concentration of 10mM for use;
(2) respectively taking 1ml of florfenicol monoclonal antibody with the concentration of 2mg/ml and 1ml of trimethoprim monoclonal antibody with the concentration of 2mg/ml into a 2ml centrifugal tube, adding 27 mu l of biotin prepared in the previous step into each centrifugal tube, uniformly mixing by vortex, and reacting for 30min on a rotary incubator;
(3) transferring the mixture to an ultrafiltration tube after the reaction is finished, centrifuging the mixture for 15min at 12000g, and discarding waste liquid;
(4) adding 2ml of PBS buffer solution into the ultrafiltration tube, 12000g, centrifuging 15ml, and discarding waste liquid;
(5) repeating the step (4) for 5 times, re-dissolving 200 mu l of PBS, collecting the labeled antibody, and measuring the concentration by using a BCA kit.
The method for preparing the streptavidin fluorescent microspheres comprises the following steps:
(1) adding 1mL of marking buffer solution 0.05M MES buffer solution into a 2mL centrifuge tube, adding 1mg of fluorescent microspheres, and uniformly mixing by vortex; adding 4 μ l NHS (25 mg/ml) and 4 μ l EDC (25 mg/ml), mixing by vortex, and reacting for 20min on a rotary incubator;
(2) centrifugally separating the activated fluorescent microspheres, discarding supernatant, taking precipitate, adding 1000 μ l of borate buffer solution, performing ultrasonic treatment for 2s, performing intermittent treatment for 5s, and repeating the steps for 5 times to break up the microspheres;
(3) adding 100 mu g of streptavidin into the activated microspheres, uniformly mixing by vortex, and placing on a rotary incubator for reaction for 60 min;
(4) adding 100 μ l of 10% casein blocking solution into each tube, mixing uniformly by vortex, placing on a rotary incubator after ultrasonic treatment, sealing for 60min, centrifuging, removing supernatant, and collecting precipitate;
(5) adding 1mL of microsphere preservation solution into the precipitate, fully and uniformly mixing, carrying out centrifugal separation, sucking supernatant, and taking the precipitate;
(6) repeating the step (5) for 2-3 times to fully remove unreacted streptavidin; then adding 1mL of microsphere storage solution, and fully and uniformly mixing for later use.
The preparation process of the florfenicol detection microsphere and the trimethoprim detection microsphere is as follows
(1) Adding 5 mu g of long-chain biotinylated antibody into 1ml of streptavidin fluorescent microspheres, uniformly mixing by vortex, and reacting for 30min on a rotary incubator;
(2) after the reaction is finished, centrifuging at 16500rpm for 20min, sucking the supernatant, and taking the precipitate;
(3) adding 1mL of microsphere preservation solution, mixing well, centrifuging at 16500rpm for 20min, and removing supernatant;
(4) repeating the step (3) for 3 times, and fully removing the unreacted biotin-labeled antibody;
(5) adding 1mL of microsphere preservation solution into the precipitate, and fully and uniformly mixing for later use;
the conjugate pad was prepared by the following steps: mixing florfenicol detection microspheres, trimethoprim detection microspheres and goat anti-chicken IgY polyclonal antibody marking microspheres to enable the final concentration of the microspheres to be 0.2mg/ml, 0.1mg/ml and 0.01mg/ml respectively, carrying out ultrasonic treatment in an ultrasonic cleaning instrument for 2min, spraying the microspheres onto a glass fiber membrane 8964 pad with the width of 1.2cm according to 4 mul/cm and 20KPa, and drying at 45 ℃.
Preparation of test paper strip
After the sample pad, the combination pad and the reaction membrane are prepared according to the method, one side of the nitrocellulose membrane is tightly pressed with the edge of the nitrocellulose membrane for about 1-2mm to be pasted with the absorbent paper pad, the other side of the nitrocellulose membrane is tightly pressed with the edge of the nitrocellulose membrane for about 1-2mm to be pasted with the combination pad, and the other side of the combination pad is tightly pressed with the edge of the combination pad for about 1-2mm to be pasted with the sample pad. Then cutting the test strip into test strips with the width of 3.95mm, and placing the test strips into the test strip grooves of the lower cover of the detection card, wherein the upper cover of the detection card is covered to form a complete detection card.
Example 2
The embodiment provides a method for synchronously detecting the content of florfenicol and trimethoprim in poultry eggs, which specifically comprises the following steps:
(1) firstly, preprocessing a sample, and specifically comprises the following steps: taking 2g of homogenized poultry eggs which do not contain florfenicol and trimethoprim and are detected by high performance liquid chromatography as negative control substances, adding 3ml of ethyl acetate (containing 1wt% of trichloroacetic acid) into each tube, manually shaking for 30s at 4000rpm, and centrifuging for 1 min. Taking 1ml of supernatant into a 15ml centrifuge tube, adding 5ml of n-hexane and 0.5ml of re-solution, standing for layering, and taking the lower-layer water phase as a solution to be detected;
(2) and adding the liquid to be detected extracted in the pretreatment into a sample hole of the detection card, horizontally standing for 10min, and then inserting the liquid into a fluorescence immunochromatographic instrument to read the result. The results were included in the standard set prepared in example 3, and the florfenicol and trimethoprim content of the sample was calculated.
Example 3
Preparation of test standards
Preparing a series of standard products: adding 16 mu l of florfenicol standard substance (10 mg/kg) and 16 mu l of trimethoprim standard substance (10 mg/kg) into 968 mu l of methanol solution, uniformly mixing to obtain a mixed standard substance 1 (the florfenicol is 160 mu g/kg, the trimethoprim is 160 mu g/kg), and then diluting for 5 times in a multiple ratio to obtain a mixed standard substance 2, a mixed standard substance 3, a mixed standard substance 4, a mixed standard substance 5 and a mixed standard substance 6; taking 7 15ml centrifuge tubes, adding 2ml blank sample without florfenicol and trimethoprim into each tube, adding 50 ul of mixed standard substance 1-6 into 6 tubes, adding 50 ul of methanol into the 7 th tube, and mixing uniformly.
The florfenicol concentration gradient is: 4. mu.g/kg, 2. mu.g/kg, 1. mu.g/kg, 0.5. mu.g/kg, 0.25. mu.g/kg, 0.125. mu.g/kg, 0. mu.g/kg;
the trimethoprim concentration gradient is as follows: 4. mu.g/kg, 2. mu.g/kg, 1. mu.g/kg, 0.5. mu.g/kg, 0.25. mu.g/kg, 0.125. mu.g/kg, 0. mu.g/kg;
adding 3ml of the extracting solution into each tube, manually shaking for 30s at 4000rpm, centrifuging for 2min, taking 1ml of supernatant, adding into a 15ml centrifuge tube, adding 5ml of n-hexane and 0.5ml of complex solution, manually shaking for 10s, and standing for layering. Adding 100 μ l of lower water phase into the detection card, standing for 10min, and then inserting into a fluorescence immunochromatography instrument for reading to obtain the ratio (T/C) of the detection line and the quality control C line. And (3) drawing a working curve by taking the concentration gradient as an abscissa and the corresponding T/C value as an ordinate:
the standard curve for florfenicol detection is:
y=3.95762/(1+(x/0.11053)^0.93504)+0.16273 R2=0.99994
linear range: 0.1-4 mug/kg
The standard curve for trimethoprim detection was: y = -1.23982-2.17326 a x R2=0.99728
Linear range: 0.2-4 mug/kg
Example 4
Minimum detection limit:
taking negative samples, repeating the steps for 10 times, calculating the T/C mean value and the Standard Deviation (SD), then subtracting 2 SD from the mean value, substituting the obtained value into a dose-response curve, and obtaining the florfenicol sensitivity of the method to be 0.05 mu g/kg; the sensitivity of trimethoprim is 0.1. mu.g/kg.
Relative standard deviation:
adding the standard substance into the negative sample until the final concentration of florfenicol is 0.2 mug/kg, 0.4 mug/kg and 1.6 mug/kg; the final concentration of trimethoprim is 0.25. mu.g/kg, 0.5. mu.g/kg, 2. mu.g/kg. The test was repeated 10 times per concentration and the Coefficient of Variation (CV) of the test concentration was calculated. The results are shown in table 1, and therefore, the coefficient of variation of the detection is less than 15%, and the detection method has high precision and meets the requirement of conventional rapid detection.
TABLE 1 relative standard deviation of florfenicol, trimethoprim at different concentrations
And (3) standard addition recovery rate:
adding the standard substance into the negative sample until the final concentration of florfenicol is 0.2 mug/kg, 0.4 mug/kg and 1.6 mug/kg; the final concentration of trimethoprim is 0.25. mu.g/kg, 0.5. mu.g/kg, 2. mu.g/kg. The test was repeated 10 times per concentration, and the recovery was calculated by dividing the test concentration by the spiked concentration. The results are shown in table 2, from which it can be seen that the present detection method has a higher accuracy.
TABLE 2 recovery of florfenicol and trimethoprim from the addition of standard
In order to prove and verify the technical scheme provided by the application, an application example of the technology provided by the application is given below.
Application example 1
Detection of different kinds of samples
Taking 5 standard adding samples of eggs of chicken, ducks, geese, pigeons and quail, wherein the standard adding final concentration of florfenicol is 0.2 mu g/kg, and the standard adding final concentration of trimethoprim is 0.25 mu g/kg. Taking 10 tubes and 2 ml/tube of each sample, adding 3ml of extracting solution into each tube, manually shaking for 30s at 4000rpm, centrifuging for 2min, taking 1ml of supernate, adding into a 15ml centrifuge tube, adding 5ml of n-hexane and 0.5ml of complex solution, manually shaking for 10s, standing and layering. Adding 100 μ l of lower water phase into the detection card, standing for 10min, inserting into a fluorescence immunochromatography instrument for reading to obtain the ratio (T/C) of two detection lines to a quality control C line, substituting the T/C value into a standard curve to obtain the feedback concentration, and further calculating the standard addition recovery rate and the repeatability. The results are shown in Table 3.
TABLE 3 results of standard recovery and repeatability experiments of florfenicol and trimethoprim in different samples
The recovery rate of various samples or different samples based on the matrix difference is different, and the overall recovery rate and repeatability can meet the requirement of on-site rapid detection.
Claims (10)
1. An immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in poultry eggs comprises a bottom plate, wherein a water absorption paper pad, a reaction membrane, a combination pad and a sample pad are sequentially connected to the bottom plate from left to right, and is characterized in that the reaction membrane is marked with three lines of a florfenicol detection line T1, a trimethoprim detection line T2 and a quality control C line, and the combination pad is coated with florfenicol detection microspheres, trimethoprim detection microspheres and goat anti-chicken IgY polyclonal antibody labeled microspheres;
the florfenicol detection microsphere is prepared by combining a long-chain biotinylation florfenicol monoclonal antibody connected with a polyethylene glycol long-chain lengthened arm-dPEG 24-and a streptavidin fluorescent microsphere through biotin and streptavidin;
the trimethoprim detection microsphere is prepared by combining a long-chain biotinylated trimethoprim monoclonal antibody connected with a polyethylene glycol long-chain lengthened arm-dPEG 24-and a streptavidin fluorescent microsphere through biotin and streptavidin.
2. The immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs according to claim 1, wherein the reaction membrane is prepared by the following steps:
(1) diluting chicken IgY to 0.1mg/mL by using 0.01M phosphate buffer solution with 3% sucrose sugar and pH7.4 to serve as quality control C-line working solution;
(2) diluting the florfenicol coupling antigen to 0.2mg/mL by using a phosphate buffer solution of 0.01M with pH7.4 and containing 3% sucrose sugar to be used as a working solution of a florfenicol detection line;
(3) diluting the trimethoprim coupling antigen to 0.2mg/mL by using a phosphate buffer solution of 0.01M and pH7.4 containing 3% sucrose sugar, and using the diluted sample as a working solution of a trimethoprim detection line;
(4) scribing three lines of a florfenicol detection line T1, a trimethoprim detection line T2 and a quality control line C on a nitrocellulose membrane by using a gold spraying membrane scribing instrument, wherein the distance between two adjacent lines is 5mm, and the scribing concentration is 1 mul/cm; after finishing the scraping, the paper is dried in a drying oven at 45 ℃ for 48 hours.
3. The immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs according to claim 1, wherein the sample pad is prepared by the following steps:
(1) preparing 0.02M phosphate buffer solution of pH7.4 containing 1% casein, 0.15M NaCl, 1% PEG 8000 and 1.5% Triton X-100 as a treatment solution;
(2) treating fluid prepared in step (1) at a concentration of 50. mu.l/cm2Uniformly coating the mixture on glass fiber RB65, and drying for 16h at 45 ℃.
4. The immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs according to claim 1, wherein the combination pad is prepared by the following steps: mixing florfenicol detection microspheres, trimethoprim detection microspheres and goat anti-chicken IgY polyclonal antibody marking microspheres, uniformly spraying onto a glass fiber membrane pad with the width of 1.2cm, and drying at 45 ℃.
5. The immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs according to claim 1, wherein the goat anti-chicken IgY polyclonal antibody labeled microsphere is prepared by the following method: :
(1) adding 1mL of 0.05M MES buffer solution into a 2mL centrifuge tube, adding 1mg of fluorescent microspheres, and uniformly mixing by vortex; adding 4 mul of NHS with the concentration of 25mg/ml and 4 mul of EDC with the concentration of 25mg/ml into the centrifuge tube, uniformly mixing by vortex, and reacting for 20min on a rotary incubator;
(2) centrifugally separating the activated fluorescent microspheres, removing supernatant, taking precipitate, adding 1000 mu l of 0.02M borate buffer solution with pH8.0, and fully and uniformly mixing;
(3) adding 100 μ g goat anti-chicken IgY, mixing uniformly by vortex, placing on a rotary incubator, and reacting for 60 min;
(4) adding 100 μ l of 10% casein blocking solution into each tube, mixing uniformly by vortex, placing on a rotary incubator after completing ultrasonic treatment, and sealing for 60 min; and centrifuging again, sucking supernatant, retaining precipitate, adding 1mL of microsphere preservation solution, and mixing completely for later use.
6. The immunofluorescence chromatography detection card for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs according to claim 1, wherein the preparation process of the florfenicol detection microspheres and the trimethoprim detection microspheres is divided into three steps:
the first step is as follows: the preparation method of the long-chain biotinylation florfenicol monoclonal antibody and the long-chain biotinylation trimethoprim monoclonal antibody comprises the following steps:
(1) taking out the long-chain Biotin NHS-dPEG24-Biotin, balancing for 30min at room temperature, and dissolving with DMSO to a final concentration of 10mM for later use;
(2) respectively taking 1ml of florfenicol monoclonal antibody with the concentration of 2mg/ml and 1ml of trimethoprim monoclonal antibody with the concentration of 2mg/ml into a 2ml centrifuge tube, adding 27 mu l of Biotin NHS-dPEG24-Biotin prepared in the previous step into each tube, uniformly mixing by vortex, and reacting for 30min on a rotary incubator;
(3) transferring the mixture to an ultrafiltration tube after the reaction is finished, centrifuging the mixture for 15min at 12000g, and discarding waste liquid;
(4) adding 2ml of PBS buffer solution into the ultrafiltration tube, 12000g, centrifuging 15ml, and discarding waste liquid;
(5) repeating the step (4) for 5 times, redissolving by 200 mu l of PBS, collecting the labeled antibody, and measuring the concentration by using a BCA kit;
the second step is that: the preparation method of the streptavidin fluorescent microsphere comprises the following steps:
(1) adding 1mL of marking buffer solution 0.05M MES buffer solution into a 2mL centrifuge tube, adding 1mg of fluorescent microspheres, and uniformly mixing by vortex; adding 4. mu.l NHS with concentration of 25mg/ml and 4. mu.l EDC with concentration of 25mg/ml, mixing uniformly by vortex, and reacting for 20min on a rotary incubator;
(2) centrifugally separating the activated fluorescent microspheres, removing supernatant, taking precipitate, adding 1000 mu l of borate buffer solution, and fully and uniformly mixing;
(3) adding 100 mu g of streptavidin into the activated microspheres, uniformly mixing by vortex, and placing on a rotary incubator for reaction for 60 min;
(4) adding 100 μ l of 10% casein blocking solution into each tube, mixing uniformly by vortex, placing on a rotary incubator after ultrasonic treatment, sealing for 60min, centrifuging, removing supernatant, and collecting precipitate;
(5) adding 1mL of microsphere preservation solution into the precipitate, fully and uniformly mixing, carrying out centrifugal separation, sucking supernatant, and taking the precipitate;
(6) repeating the step (5) for 2-3 times to fully remove unreacted streptavidin; adding 1mL of microsphere preservation solution, and fully and uniformly mixing for later use;
the third step: the preparation process of the florfenicol detection microsphere and the trimethoprim detection microsphere comprises the following steps:
(1) respectively adding 5 mu g of each long-chain biotinylation florfenicol monoclonal antibody and long-chain biotinylation trimethoprim monoclonal antibody into 2 tubes of fluorescent microspheres containing 1ml of streptavidin, uniformly mixing in a vortex manner, and reacting for 30min on a rotary incubator;
(2) after the reaction is finished, centrifuging at 16500rpm for 20min, sucking the supernatant, and taking the precipitate;
(3) adding 1mL of microsphere preservation solution, mixing well, centrifuging at 16500rpm for 20min, and removing supernatant;
(4) repeating the step (3) for 3 times, and fully removing the unreacted biotin-labeled antibody;
(5) adding 1mL of microsphere storage solution, and mixing well for later use.
7. A method for synchronously detecting the content of florfenicol and trimethoprim in poultry eggs is characterized by comprising the following steps:
pretreating a sample to obtain a solution to be detected;
(II) adding a solution to be detected into the detection card of claim 1, horizontally standing for 10min, and then inserting into a fluorescence immunochromatographic instrument to read the result;
thirdly, the result is brought into the standard curve, and the content of the florfenicol and the trimethoprim in the sample can be calculated;
wherein, the pretreatment method in the step (one) comprises the following steps:
(1) adding the extract into the fully homogenized egg to-be-detected liquid, manually shaking and uniformly mixing for extraction, centrifuging and taking supernatant;
(2) adding n-hexane and a redissolution into the supernatant, shaking, standing and layering to obtain a lower water phase as a solution to be detected;
the volume ratio of the poultry egg sample to be detected to the extracting solution is as follows: 2: 2-3.
8. The method for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs as claimed in claim 7, wherein the extracting solution is ethyl acetate containing 0.5-1.5wt% of trichloroacetic acid.
9. The method for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs according to claim 7, wherein the complex solution is PBS buffer solution containing 0.15M NaCl and 0.05M PB, and the pH value is 6.6.
10. The method for synchronously detecting the content of florfenicol and trimethoprim in the poultry eggs according to claim 7, wherein the volume ratio of the supernatant to the n-hexane to the complex solution is as follows: 1:5:0.5-1.
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