Qualitative detection method for content of beta-receptor stimulants in milk

A receptor agonist, qualitative detection technology, applied in the direction of biological testing, material inspection products, etc., can solve the problem that the content of β-receptor agonist needs to be improved

Active Publication Date: 2016-11-23

INNER MONGOLIA MENGNIU DAIRY IND (GRP) CO LTD

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AI-Extracted Technical Summary

Problems solved by technology

[0003] However, the current detection methods for the c...

Abstract

The invention discloses a qualitative detection method for the content of beta-receptor stimulants in milk. The qualitative detection method includes the steps that 1, a substrate, a positive quality control product and to-be-detected milk are detected respectively with the enzyme-linked immunoassay method, the OD value of the substrate, the OD value of the positive quality control product and the OD valued of the to-be-detected milk are obtained, wherein the positive quality control product is reconstituted milk containing the known-content beta-receptor stimulants, the substrate is reconstituted milk which is free of beta-receptor stimulants, and the reconstituted milk is obtained in the mode that whole milk powder purchased from New Zealand is dissolved in water; 2, based on the detection result, the to-be-detected milk is subjected to qualitative detection. The qualitative detection method is easy and convenient to operate and high in accuracy.

Application Domain

Biological testing

Technology Topic

Enzyme linked immunoassayChemistry +4

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Examples

Experimental program(3)
Comparison scheme(1)

Example Embodiment

[0021] Example 1

[0022] 1. Reagents and solutions.

[0023] β-receptor agonist detection kit, purchased from EP company in the Netherlands.

[0024] This substrate: reconstituted milk, obtained by dissolving whole milk powder purchased from Fonterra Company in New Zealand in water at a mass ratio of 1:7.

[0025] Positive quality control product: reconstituted milk containing known content of β-agonist.

[0026] 2. Equipment and instruments.

[0027] BioTek's ELx808 microplate reader has a detection wavelength of 450nm.

[0028] Waters premier ultra performance liquid chromatography tandem mass spectrometer.

[0029] 3. Operation steps.

[0030] (1) Take out the required number of microwell tubes and insert them into the microwell plate, and record the position of the microwells;

[0031] (2) Centrifuge the substrate, the positive quality control product, and the milk to be tested at a temperature of 2 to 8°C and a rotation speed of 3000 g for 10 minutes, and collect the supernatant, and add the supernatant to the microporous tube. , 50μL per well;

[0032] (3) Add 25μL of enzyme conjugate to the microporous tube containing the substrate, the positive quality control product and the milk to be tested;

[0033] (4) Add 25 μL of antibody solution to the microporous tube containing the enzyme conjugate;

[0034] (5) Seal the plate with glue strips, shake the plate for 1 min, and incubate in the dark at 2°C-8°C for 1 hour;

[0035] (6) After emptying the wells of the microplate upside down, shake it down steadily and quickly, and place the microplate upside down on absorbent paper and tap to remove the liquid in the tube. Use a cleaning solution (250μL/well). ) Inject into the microporous tube, mix and pour the liquid in the microporous tube again, repeat the above operation 3 times;

[0036] (7) Add 100 μL of the substrate solution to the microporous tube containing the antibody solution, and incubate in the dark at room temperature (20-25°C) for 30 minutes;

[0037] (8) Add 100μL of stop solution to the microporous tube containing the substrate solution, and immediately read the absorbance at 450nm wavelength;

[0038] (9) Based on the following criteria, qualitatively determine the milk to be tested:

[0039] (a) If the OD value of the milk to be tested is less than the OD value of the positive quality control product, the content of β-agonist in the milk to be tested is greater than the content of β-agonist in the positive quality control product;

[0040] (b) If the OD value of the milk to be tested is greater than or equal to the OD value of the positive quality control product, the content of β-agonist in the milk to be tested is less than or equal to the content of β-agonist in the positive quality control product; or

[0041] (c) If the OD value of the substrate is less than the OD value of the positive quality control, the test result is invalid.

Example Embodiment

[0042] Example 2

[0043] Accuracy test

[0044] Take 4 kinds of milk samples without β-agonist, add different concentrations of β-agonist standard products (commercially available) to them according to the concentration shown in the table below to obtain spiked milk, and use them separately The method of Example 1 and GB/T22965-2008 "Determination of 12 β-stimulant Residues in Milk and Milk Powder Liquid Chromatography-Tandem Mass Spectrometry" (hereinafter referred to as chromatographic method) carried out two parallel detections on different spiked milk, The substrate is the reconstituted milk without β-agonist, and the positive quality control product is the reconstituted milk with 0.15ng/mL β-agonist.

[0045] The results are shown in Table 1. Add β-agonist standard products to the four milk samples. When the concentration is lower than 0.15ng/mL, the absorbance value is higher than the positive quality control product (average OD value is 0.916), and all can be qualitatively judged as negative ; When the concentration is ≥0.15ng/mL, the absorbance value is lower than the positive quality control product, and it can be qualitatively judged as positive. The sample added with the standard was tested by chromatography to confirm the concentration of the standard addition, which proved the accuracy of this experiment.

[0046] Table 1 Accuracy Test

[0047]

[0048]

[0049] Note: Positive means that the β-agonist content in the sample is greater than or equal to 0.15ng/mL, and negative means that the sample does not contain or the β-agonist content is less than 0.15ng/mL.

Example Embodiment

[0050] Example 3

[0051] False positive rate detection

[0052] Take 4 kinds of milk samples without β-agonist, add different concentrations of β-agonist standard products (commercially available) to them according to the concentration shown in the table below to obtain spiked milk, and use them separately The method of Example 1, the enzyme-linked immunoassay kit and the chromatographic method were used to perform two parallel detections on different spiked milk. The substrate was the reconstituted milk without β-receptor agonist, and the positive quality control was 0.15ng. /mL β-agonist reconstituted milk.

[0053] The detection process of the ELISA kit is as follows:

[0054] (1) Measure the OD value of the sample to be tested according to steps (1) to (8) of Example 1, wherein there is no need to set the substrate and positive quality control material in step (1);

[0055] (2) Measure the OD value of different concentrations of β-receptor agonist standard products according to the steps (1) to (8) of Example 1 (wherein step (1), there is no need to set the substrate and positive quality control products), and draw Standard curve of β-agonist concentration-OD value;

[0056] (3) Substitute the OD value determined in step (1) into the standard curve to obtain the β-receptor agonist content in the sample to be tested.

[0057] The results are shown in Table 2. Among them, the OD detected by the method in Example 1 450 In the two columns of data, the data in the left column is the test result of adding 0ng/mL β-agonist to the sample, and the data in the right column is the test result of adding 0.15ng/mL β-agonist to the sample. The meanings of the two columns of data in the determination of the method in Example 1, the test result of the kit, and the test result of the chromatogram method are the same as the above description, and will not be repeated here.

[0058] It can be seen that for 20 milk samples of different batches, when the ELISA kit method was used, the results of 7 were higher than 0.15ng/mL, while the results of the chromatographic method were all <0.1ng/mL is a false positive result, and the false positive rate is 35%. The main reason may be that the composition of the sample to be tested is more complicated, which interferes greatly with the reagents, resulting in a high false positive phenomenon. However, the background test results detected by the method of Example 1 are all negative, and the false positive rate is 0%, which proves that this method can effectively reduce the false positive detection of β-agonist in the finished milk under the premise of ensuring the accuracy of the test results. The positive rate achieves the effect of saving testing costs.

[0059] Table 2 False positive rate detection

[0060]

[0061] Note: Positive means that the β-agonist content in the sample is greater than or equal to 0.15ng/mL, and negative means that the sample does not contain or the β-agonist content is less than 0.15ng/mL.

PUM

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