Introduction to the detection method of zearalenone in cereals
4 equipment and reagents needed
4.1 Instrument: CSY-E96C zearalenone rapid detector, printer, homogenizer, oscillator, centrifuge, graduated pipette, balance (sensitivity 0.01g)
4.2 Micropipette: single channel 20μl-200μl, 100μl-1000μl, multi-channel 300μl
4.3 Reagents: acetonitrile, deionized water
5 sample pretreatment
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: sample extract
90% acetonitrile, ie V acetonitrile: V deionized water = V9: V1.
5.3 Sample pre-processing steps:
5.3.1 Cereals (low fat content of rice, corn, millet, etc.) and compound feed
Approach
1) Weigh 2 g of the pulverized sample in a 50 ml centrifuge tube, add 8 ml of the sample extract, shake for 5 minutes, and centrifuge at room temperature for 4000 rpm for 10 minutes;
2) Take 0.5ml of supernatant, add 2ml of deionized water, and mix;
3) Take 50 μl for analysis.
Dilution multiple: 20 detection limit: 6ppb
6 enzyme-linked immunoassay steps
Remove the required reagent from the 4 ° C refrigerated environment, and equilibrate at room temperature for more than 30 min. When the washing solution is refrigerated, the crystal may be returned to room temperature to be fully dissolved. Each liquid reagent should be shaken before use. Remove the required number of microplates and frames, place the unused microplates in a ziplock bag and store at 2-8 °C.
Before the start of the experiment, the 20x concentrated washing solution was diluted with deionized water to a working washing solution at 1:19 (i.e., 1 part concentrated washing solution + 19 parts deionized water).
6.1 No.: The micropores corresponding to the sample and the standard are numbered sequentially, and each sample and the standard are made in parallel with 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 the respective microwells, 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 30 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: 50 μl of substrate A was added to each well, and 50 μl of substrate B was added. The mixture was gently shaken for 5 seconds, and mixed at 25 ° C for 15 minutes.
6.5 termination: 50 μl of stop solution was added to each well, and the mixture was gently shaken to terminate the reaction.
6.6 Measured absorbance: The absorbance value per well was measured at 450 nm using a CSY-E96C zearalenone detector (double wavelength 450/630 nm is recommended). The assay should be completed within 10 minutes of terminating the reaction.
7 results analysis
7.1 Calculation of percent absorbance
The percent absorbance of the standard solution or sample is equal to the average value of the absorbance value of the standard solution or sample (double well) divided by the absorbance value of the first standard solution (0 ppb), multiplied by 100%, ie
Percent 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 standard curve
The semi-logarithmic plot of the standard solution is plotted with the percent absorbance of the standard solution as the ordinate and the logarithm of the corresponding standard solution concentration (ppb) as the abscissa. 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.
If the kit professional analysis software is used for calculation, it is more convenient for accurate and rapid analysis of a large number of samples. (Welcome to Shenzhen Fenyi Instrument Manufacturing Co., Ltd.)
8 considerations
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.
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