1. Sample pretreatment
Grind a representative sample of 2kg to pass through a 20-mesh sieve with more than 95% of the sample. Mix well for sampling.
Prepare the extraction solution (70% methanol): Mix methanol with distilled water according to a volume ratio of 7:3 according to the amount used.
Weigh 10g ± 0.1g of the above sample into a clean covered container, and add 50mL of 70% methanol (the ratio of the sample to 70% methanol is 1:5 (w: v)).
Fully oscillate and extract for 3 minutes, let it stand and take the supernatant for centrifugation at 4000rpm for 5 minutes. Take the supernatant for testing.
2. Sample detection
Premixing: Take an appropriate amount of pre-mixed strip according to the need, add 60 µL of the calibration product/measured sample to the micropores and record its position. Then add 120 µL of the enzyme-labeled reagent to each hole, and mix well by pipetting or horizontal shaking. (The pre-mixing ratio of the calibration product/measured sample to the enzyme-labeled reagent is 1:2. For replicate holes, 100 µL of the calibration product/measured sample can be mixed with 200 µL of the enzyme, and 100 µL can be added to each of the two detection plate holes respectively.)
Sample addition: Add 100 µL of the pre-mixed liquid to the detection plate hole. Cover with the cover membrane and incubate at 37°C for 10 minutes.
Plate washing: Shake off the solution in the micropores, fill with distilled water, repeat 4 times, and pat dry on the absorbent paper.
Chromogenic: Add 100 µL of the chromogenic solution to each hole. Cover with the cover membrane and incubate at 37°C for 5 minutes.
Termination: Add 100 µL of the termination solution to each hole, and read the absorbance value at 450/630nm double wavelengths immediately.
Experimental operation tips
Doing replicate holes for the calibration product and the measured sample can improve the accuracy of thedetection.
Replace the pipette tip when drawing different reagents to prevent cross-contamination.
When using the pipette, be careful to avoid drawing in bubbles and affecting the volume of the added liquid. The way of drawing once and dispensing twice is easy to splatter after dispensing the liquid, so it is recommended to use the way of drawing twice and dispensing once to add the liquid vertically to the center of the micropores.
The liquid should be dispensed quickly and should not form water droplets.
The transfer of the mixed liquid in the pre-mixing plate to the detection plate starts the reaction. To reduce the error, please control the time difference between the transfer of different micropores (increase the transfer speed,control the number of single-sample tests. It is better to use a multi-channel pipette to test 4 strips at a time;it is better to use a single-channel pipette to test 2 strips at a time).
Do not use expired kits, and do not mix different batches of reagents.
Please keep the kit properly. Freezing, room temperature, and high temperature conditions will accelerate the aging of the kit. In use, please avoid cross-contamination of different reagents. If an abnormality of the reagent is found, such as the chromogenic solution turning blue, or the reagent being turbid, do not use it.
This method is only for the rapid preliminary screening of samples. For confirmation, please refer to methods such as liquid chromatography.
