Aquatic product furazolidone metabolite detector operation mode: Nitrofurans are broad-spectrum antibiotics that kill most Gram-positive and Gram-negative bacteria, fungi, and protozoa. They act on the microbial enzyme system, inhibit acetyl-CoA, interfere with the metabolism of microbial sugars, and thus inhibit bacteria. Nitrofurans have been widely used in livestock and aquaculture to treat enteritis, acne, red fin disease, ulcer disease caused by Escherichia coli or Salmonella. Due to the carcinogenic and teratogenic side effects of nitrofuran drugs and their metabolites on the human body, the Chinese Ministry of Health listed the nitrofuran drugs furazolidone, furantrone, nitrofurantoin and nitrofurazone on March 22, 2010. Blacklist of non-edible substances. Nitrofurans are commonly found in the following four types: furazolidone, furanone, nitrofurantoin, and nitrofurazone. Nitrofurans are all yellow powders with stable properties and are odorless or slightly bitter. Furazolidone is hardly soluble in water and ethanol, nitrofurazone is hardly soluble in water, slightly soluble in ethanol, and nitrofurantoin is almost insoluble in water and slightly soluble in ethanol. The nitrofuran prototype drug is rapidly metabolized in the living body, and its metabolites are AOZ, AMOZ, AHD, SEM, and protein binding are quite stable. Therefore, the detection of metabolites is often used to reflect the residual state of nitrofuran drugs. Required equipment and reagents 5 sample pretreatment 6 Enzyme-linked immunoassay procedure Take the required reagents from the 4 ° C refrigerated environment and equilibrate at room temperature for more than 30 min. When the washing liquid is refrigerated, there may be crystallization to return to room temperature to fully dissolve. Each liquid reagent must be shaken before use. uniform. Remove the required number of microplates and frames, place the unused microplates in a ziplock bag and store at 2-8 °C. 7 Results analysis 8 Precautions Hidden Safe Box,Bedroom Safe Box,Intelligent Safe Box,Cash Box Hebei Yingbo Safe Boxes Co.,Ltd , https://www.ybsafebox.com
4.1 Instrument: CSY-E96S aquatic product drug residue detector , homogenizer, nitrogen blow dryer, oscillator, centrifuge, graduated pipette, balance (sensitivity 0.01g)
4.2 Micropipette: single channel 20μl-200μl, 100μl-1000μl, multi-channel 300μl
4.3 Reagents: ethyl acetate, n-hexane, sodium hydroxide, concentrated HCl, K2HPO4•3H2O, potassium nitroferrocyanide (K2Fe(CN)5(NO)▪2H2O), ZnSO4•7H2O
5.1 Notes before sample processing:
Laboratory equipment must be clean and use disposable tips to avoid contamination and interference with experimental results.
5.2 Dosing:
Solution 1: 0.36M potassium nitroferrocyanide solution (milk, milk powder sample)
11.9 g of potassium nitroferrocyanide plus deionized water was dissolved in 100 ml.
Solution 2: 1.04M zinc sulphate solution (milk, milk powder sample)
29.8 g of zinc sulfate plus deionized water was dissolved in 100 ml.
Solution 3: 0.1M K2HPO4 solution Weigh 11.4g K2HPO4â–ª3H2O with deionized water to dissolve and dissolve to 500ml.
Dosing 4:1M HCL
8.6 mL of concentrated HCL plus deionized water to a volume of 100 mL.
Dosing 5:1M NaOH solution Weigh 4g NaOH and dilute to 100ml with deionized water.
Solution 6: complex solution The 2× complex solution was diluted 2 times with deionized water for reconstitution of the sample, and the complex solution was stored at 4 ° C for one month.
5.3 Sample pre-processing steps:
5.3.1 Milk sample processing method
1) Take 5ml sample in a centrifuge tube, add 250ul potassium nitroferricyanide solution (dispensing solution 1) and mix for 30 seconds. Add 250ul zinc sulphate solution (liquid 2) and mix for 30 seconds, 15°C 4000 rpm. Centrifugation for 10 minutes;
2) Take the supernatant 1.1ml, add 4ml deionized water, 0.5 ml 1M hydrochloric acid solution and 100μl derivatization reagent, shake for 5 minutes;
3) Incubate at 37 ° C overnight (about 16 hours) or 50 ° C (more than 50 ° C will affect the stratification effect) incubation in water bath for 3 hours;
4) separately add 5 ml of 0.1 M K2HPO4 solution, 0.4 ml of 1 M NaOH solution and 5 ml of ethyl acetate, and shake for 5 minutes;
5) Centrifugation at 4000 rpm for 10 minutes at room temperature;
6) Take out 2.5 ml of the supernatant liquid to another centrifuge tube, and blow dry at 50-60 ° C under nitrogen or air;
7) Dissolve the residue with 1 ml of n-hexane, add 1 ml of reconstituted working solution and mix thoroughly for 30 seconds; centrifuge at room temperature for 4,000 rpm for 10 minutes;
8) Remove the upper n-hexane and take 50 μl of the lower layer for analysis.
Sample dilution factor is 2 Detection limit: 0.04ppb
5.3.2 Method for processing milk powder and egg powder samples
1) Weigh 1±0.05g homogenized sample in a centrifuge tube, add 4ml deionized water, 0.5ml 1M hydrochloric acid solution and 100μl derivatization reagent, shake for 5 minutes;
2) Incubate overnight at 37 ° C (about 16 hours) or 50 ° C (more than 50 ° C will affect the stratification effect) in a water bath for 3 hours;
3) Add 250ul of potassium nitroferrocyanide solution (dispensing solution 1) and shake for 30 seconds. Add 250ul of zinc sulfate solution (liquid 2) and mix for 30 seconds, and centrifuge at 1000°C for 10 minutes.
4) Take all the supernatant into another centrifuge tube, followed by “5.3.1 Milk Sample Processing Method, 4)â€
5.3.3 Honey, tissue, casing, liver, feed, egg sample processing methods
1) Weigh 1±0.05g homogenized sample in a centrifuge tube, add 4ml deionized water, 0.5ml 1M hydrochloric acid solution and 100μl derivatization reagent, shake for 5 minutes;
2) Follow-up "5.3.1 Milk sample processing method, 3)"
5.3.4 Method of processing cooked food samples
1) Weigh 1 ± 0.05g homogenate in a 50ml centrifuge tube, add 4.5ml methanol and 0.5ml deionized water, shake for 2min, room temperature 4000 rev / min, centrifuge for 5 minutes, remove all liquid;
2) Add 5 ml of acetonitrile and 5 ml of n-hexane, shake for 2 min, centrifuge at room temperature for 4000 rpm, and centrifuge for 5 minutes to remove all the liquid;
3) adding 4 ml of deionized water, 0.5 ml of 1 M hydrochloric acid solution and 100 μl of derivatization reagent to the precipitate, and shaking for 5 minutes;
Note: Please add high standards at this step for the addition and recovery test of cooked food.
4) Follow-up "5.3.1 Milk sample processing method, 3)"
Before the start of the experiment, the 20X concentrated washing solution was diluted with 20 times of deionized water into a working washing solution.
6.1 No.: The micropores corresponding to the sample and the standard are numbered sequentially. Each sample is parallel to the standard 2 holes, and the position of the standard hole and the sample hole is recorded.
6.2 Addition reaction: Add standard or sample 50μl/well to each microwell, then add 50μl/well of enzyme label, then add 50μl/well of antibody working solution, seal the plate with cover membrane, and gently shake 5 Mix in seconds and react at 25 ° C for 45 minutes.
6.3 Washing: Carefully uncover the cover film, dry the liquid in the well, and wash it thoroughly with the working washing liquid 250μl/well 5 times, each time interval 30 seconds, pat dry with absorbent paper (bubbles that have not been removed after pat drying) A clean gun puncture).
6.4 Color development: Add 50 μl of substrate solution A to each well, add 50 μl of substrate solution B, mix gently by shaking for 5 seconds, and develop color for 15 minutes at 25 ° C in the dark.
6.5 End: Add 50 μl of stop solution to each well, mix gently by shaking, and stop the reaction.
6.6 Measured absorbance: The absorbance value of each well was measured at 450 nm using a furazolidone metabolite detector of the deep fen instrument (recommended with dual wavelength 450/630 nm). The assay should be completed within 10 minutes of terminating the reaction.
7.1 Calculation of Percent Absorbance The percent absorbance of the standard or sample is equal to the average of the absorbance values ​​of the standard or sample (double well) divided by the absorbance of the first standard (0 ppb), multiplied by 100%, that is, the absorbance value (%) = A × 100%
A0
A—the average absorbance value of the standard solution or sample solution
Average absorbance value of A0—0ppb standard solution
7.2 Drawing and calculation of the standard curve The percent absorbance of the standard solution is plotted on the ordinate, and the logarithm of the corresponding standard solution concentration (ppb) is plotted on the abscissa. The semi-logarithmic plot of the standard solution is plotted. Substituting the percent absorbance of the sample into the standard curve, reading the concentration corresponding to the sample from the standard curve, and multiplying the corresponding dilution factor is the actual concentration of the analyte in the sample.
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8.1 Room temperature below 25 ° C or reagents and samples not returned to room temperature (25 ° C) will result in low OD values ​​for all standards.
8.2 If the plate hole is dry during the washing process, the standard curve will not be linear and the repeatability is not good. Therefore, the next step should be taken immediately after the plate is patted dry.
8.3 The mixing should be uniform, the washing should be thorough, and the reproducibility in the ELISA analysis depends largely on the consistency of the washing.
8.4 Seal the microplate with a cover film during all incubations to avoid light exposure.
8.5 Do not use kits that have expired. Do not exchange reagents from different batches.
8.6 If any color of the coloring solution indicates deterioration, it should be discarded. A 0 standard absorbance value of less than 0.5 units (A450nm < 0.5) indicates that the reagent may deteriorate.
8.7 The reaction stop solution is corrosive and avoids contact with the skin. 
Aquatic product furazolidone metabolite detector operation mode