Fluorescence labeling-based quantitative detection method for milk adulteration in sheep milk

Abstract

The invention discloses a fluorescence-labeled quantitative detection method for milk adulteration in sheep milk, which comprises the following steps: (1) mixing and incubating a sample to be detected and immunomagnetic beads to obtain a suspension, carrying out magnetic separation to remove uncombined antibodies, and sealing non-specific binding sites on the magnetic beads by using a BSA solution; (2) carrying out thermal denaturation on the 5-FAM labeled nucleic acid aptamer BLG-14, and then carrying out ice bath renaturation; (3) mixing and incubating the aptamer BLG-14 obtained in the step (2) and the immunomagnetic beads combined with the beta-lactoglobulin obtained in the step (1); and (4) centrifuging, taking supernate, and detecting the fluorescence intensity by using a fluorospectro photometer or a smart phone fluorescence module. According to the invention, the aptamer BLG-14 and bovine beta-lactoglobulin are subjected to high-specificity binding (delta F value is 6-7 times higher than that of other lactoproteins, Plt; 0.01), the detection limit is optimized to 0.665% by combining the targeted enrichment effect of the immunomagnetic beads, the sensitivity is close to that of laboratory-grade chromatography, and no complex equipment is needed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food safety detection, and particularly relates to a method for quantitatively detecting adulteration of cow milk in sheep milk based on fluorescent labeling. BACKGROUND

[0002] Most of the adulteration of sheep milk involves the addition of cow milk, and in this case, the detection of adulteration is very difficult. On the one hand, the appearance of cow milk and sheep milk is very similar, and it is difficult to distinguish the two with the naked eye; on the other hand, there is a lack of rapid detection methods and equipment on the market. In order to solve the problem of detecting adulterated cow milk in sheep milk, scholars at home and abroad have conducted a large number of researches and developed a variety of methods, such as chromatography, mass spectrometry, electrophoresis, PCR, immunization and near-infrared spectroscopy. Kourkouli et al. (2024) developed a specific qualitative touchdown (TD) PCR method based on the differentiation of the peak area of the melting curve after modification of the cow-specific primer to detect the amount of added cow milk in goat and sheep milk, which can identify as low as 1% of the presence of cow milk. Another study developed a method for simultaneously quantitatively detecting whey and whole milk powder in goat or sheep milk products by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) to quantify four characteristic peptides of casein and two major whey proteins. The quantification limit of the target protein is 0.01-0.05 g / 100 g. Although they are mature technologies, they cannot meet the advantages of strong specificity, high sensitivity and quantitative detection, and the detection equipment is expensive and susceptible to environmental influences. Therefore, it is particularly important to improve the separation specificity and detection sensitivity of the target.

[0003] In the prior art, the detection limit of infrared spectroscopy is usually higher than 5%, the sensitivity of PCR can reach 1%, but the operation is complex and susceptible to contamination, and the high-precision chromatography-mass spectrometry (UHPLC-MS / MS) can detect as low as 0.01%-0.05% of adulteration, but it relies on expensive equipment and cannot be applied on site. In addition, the traditional chromatography-mass spectrometry method takes 4-6 hours for single detection, with a cost of more than 100 US dollars; the PCR method takes 3-5 hours (including DNA extraction), with a cost of about 30 US dollars; and the immunization method (such as ELISA) has strong specificity, but the cost of antibodies is high and the stability is poor.

[0004] Therefore, how to obtain a method for quantitatively detecting adulteration of cow milk in sheep milk with low cost, rapidity and resistance to matrix interference is still a technical problem to be solved by those skilled in the art. SUMMARY

[0005] Based on the above reasons, the present application proposes a method for quantitatively detecting adulteration of cow milk in sheep milk based on fluorescent labeling. Specifically, in order to achieve the purpose of the present application, the present application proposes the following technical solution:

[0006] One aspect of the present application relates to a method for quantitatively detecting adulteration of sheep milk with cow milk, which comprises the following steps:

[0007] (1) mixing and incubating the sample to be tested with immunomagnetic beads to obtain a suspension, removing unbound antibodies by magnetic separation, and blocking non-specific binding sites on the magnetic beads with a BSA solution; the immunomagnetic beads are magnetic microspheres modified with anti-β-lactoglobulin antibodies;

[0008] (2) ice-bath renaturation after thermal denaturation of 5-FAM-labeled nucleic acid aptamer BLG-14; the sequence of the 5-FAM-labeled nucleic acid aptamer BLG-14 is:

[0009] FAM-5'-CGACGATCGGACCGCAGTACCCACCCACCAGCCCCAACATCATGCCCATCCGTGTGTG-3'

[0010] (3) mixing and incubating the aptamer BLG-14 of step (2) with the immunomagnetic beads bound with β-lactoglobulin obtained in step (1);

[0011] (4) taking the supernatant after centrifugation, and detecting the fluorescence intensity using a fluorescence spectrophotometer or a smartphone fluorescence module.

[0012] In a preferred embodiment of the present application, the particle size of the immunomagnetic beads is 0.1-0.2 μm.

[0013] In a preferred embodiment of the present application, the ratio of the amount of anti-β-lactoglobulin antibodies to magnetic beads is 3-5:1.

[0014] In a preferred embodiment of the present application, the immunomagnetic beads are prepared by EDC / NHS chemical coupling method, and the bovine β-lactoglobulin antibodies are covalently fixed on the surface of aminated magnetic microspheres.

[0015] In a preferred embodiment of the present application, the immunomagnetic beads are blocked with a 2% BSA solution to reduce the adsorption of non-target proteins, which can significantly improve the enrichment capacity of target proteins.

[0016] In a preferred embodiment of the present application, after mixing and incubating the sample to be tested with immunomagnetic beads to obtain a suspension, the suspension is washed 3 times with PBST buffer containing 0.05% Tween-20 to remove non-specifically bound impurities.

[0017] The present application is directed to the rapid quantitative analysis of cow milk adulteration in dairy products (sheep milk), ensuring food quality and authenticity. The present application optimizes the detection limit to 0.665% through the high specificity binding of aptamer BLG-14 with bovine beta-lactoglobulin (ΔF value is 6-7 times higher than other milk proteins, P<0.01), combined with the targeted enrichment of immunomagnetic beads, the sensitivity is close to the laboratory chromatography, and no complex equipment is needed. In addition, the linear range of the method of the present application covers 10%-100% (R 2 =0.9318), can directly detect common adulteration concentration, without multiple dilution calibration, significantly improving the detection efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 : Schematic diagram of the present application.

[0019] Figure 2 : Optimization of the amount of immunomagnetic beads.

[0020] Figure 3 : Optimization of the amount of aptamer.

[0021] Figure 4 : Specificity and sensitivity detection diagram DETAILED DESCRIPTION

[0022] In order to further understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0023] Unless otherwise specified, the reagents involved in the embodiments of the present application are all commercially available products, which can be purchased through commercial channels.

[0024] Example 1:

[0025] Reagents and equipment:

[0026] The nucleic acid aptamer BLG-14 sequence (synthesized by Shanghai Shengong Biological Engineering Co., Ltd.) is:

[0027] FAM-5'-CGACGATCGGACCGCAGTACCCACCCACCAGCCCCAACATCATGCCCATCCGTGTGTG-3'

[0028] Amino magnetic beads with a diameter of 0.1-0.2 μm (Tianjin Microbead Technology Co., Ltd.). Sheep milk was obtained from Yuansheng Farming and Breeding Co., Ltd. (Jinchang City, China); cow milk was obtained from Muye Dairy Co., Ltd. (Xianyang City, China). Fluorescence spectrophotometer RF-6000 (Shimadzu Corporation, Japan).

[0029] Step:

[0030] 1. Activation of magnetic beads: EDC / NHS method was used to activate the magnetic beads: magnetic beads (400 μL, 10 mg / mL), EDC (300 μL, 50 mg / mL), and NHS (300 μL, 50 mg / mL) were mixed to activate the magnetic beads, which were incubated at room temperature for 30 minutes and washed with PBST for 3 times. 800 μg of anti-β-lactoglobulin antibody was added with gentle stirring. The mixture was incubated at room temperature overnight, washed with PBST for 3 times, and the magnetic beads carrying the β-lactoglobulin antibody were adsorbed by a magnetic stand and a magnet, and the unbound antibody was removed. The non-specific binding sites on the magnetic beads were blocked with a 2% BSA solution at room temperature for 30 minutes, and the resulting probe was stored at 4°C for later use.

[0031] 2. Modification of proteins: The bovine β-lactoglobulin standard was diluted with sterile PBS buffer to different concentrations (1000, 100, and 10, 1 μg / L). 500 μL of the protein solution and 50 μL of the immunomagnetic beads with different concentrations were added to a centrifuge tube. After mixing, the mixture was incubated at room temperature on a rotary plate for 45 minutes. Then, the absorbance was tested by washing with PBST for 3 times.

[0032] 3. Incubation of nucleic acid aptamer: The aptamer with a concentration of 100 nmol / L was heated in a metal bath at 95°C for 5 minutes and then in an ice bath for 10 minutes. Before use, the aptamer was treated in this way to activate its three-dimensional recognition conformation. Then, 300 μL of the suspension in step 2 above was combined with 100 μL of the aptamer as the experimental group. In addition, 300 μL of 2x binding buffer was used instead of the suspension to combine with 100 μL of the aptamer as the control group. Each of the experimental and control groups had 3 replicates. The experimental and control groups were incubated at 25°C on a 200 r / min shaker for 1 hour, and then centrifuged at 6000 r / min to obtain the supernatant. The fluorescence intensity of the experimental and control groups was measured by Qubit 3.0 fluorescence quantification instrument. The fluorescence value of β-lactoglobulin binding = average fluorescence value of the control group - average fluorescence value of the experimental group.

[0033] Table 1 Results of standard addition recovery experiment of different concentrations of milk adulteration samples

[0034]

[0035] The milk and sheep milk were mixed at 10%, 20%, 40%, 60%, 80% volume ratio, vortexed for 5 min to ensure uniform mixing, and then subjected to defatting treatment. The fat layer was removed by centrifugation at 4°C (8,000 rpm, 20 min), and the middle layer whey was collected for detection. Three parallel samples were prepared for each concentration point, and stored at 4°C for standby. The results showed that the recovery rate was 95.67%-101.07% (see Table 1), which was close to 100% and within the range of 90%-110% required by the standard methodology. Among them, the recovery rate of 10% adulteration was 97.62%, indicating that the method could still accurately capture the target protein when the concentration of adulteration was low. The recovery rate of 40% adulteration was 101.07%, which was slightly higher than the theoretical value, which may be related to the small error in fluorescence signal detection, but the overall was still within the acceptable range. The data proved that the method had good accuracy for the quantitative detection of bovine β-lactoglobulin in actual samples, and there was no significant systematic error.

[0036] In view of the key influence of the protein loading capacity of the surface of the immunomagnetic beads on the fluorescence signal intensity and detection accuracy, the concentration of bovine β-lactoglobulin was fixed at 4 mg / mL, and the concentration of the aptamer was fixed at 100 nmol / L, and the effect of the amount of immunomagnetic beads (50-250 μg) on the detection signal was investigated. Figure 2 As shown in the figure, when the amount of immunomagnetic beads increased from 50 μg to 200 μg, the fluorescence intensity showed a significant increasing trend, from 502 RFU to 1520 RFU, indicating that the specific binding efficiency of the coupling sites on the surface of the magnetic beads and the aptamer-fluorophore increased with the increase of the amount of the magnetic beads. When the amount of the magnetic beads was further increased to 250 μg, the fluorescence intensity only increased slightly to 1588 RFU, with a decrease of 78% compared with the previous stage, reflecting that the active sites on the surface of the magnetic beads were close to saturation.

[0037] In view of the need to balance signal gain and cost control for the concentration of the aptamer, under the conditions of fixed β-lactoglobulin concentration of 4 mg / mL and immunomagnetic bead amount of 200 μg, the dose-effect relationship of the volume of 100 nmol / L aptamer solution (20-120 μL) on the fluorescence signal was investigated. As shown in the figure, Figure 3 When the volume of the aptamer increased from 20 μL to 100 μL, the fluorescence intensity of the system showed an exponential growth trend, from 313 RFU to 1523 RFU, reflecting that the specific binding of the aptamer and the coupled protein on the magnetic beads increased with the increase of the effective concentration of the aptamer. When the volume of the aptamer was further increased to 120 μL, the fluorescence intensity only increased slightly to 1540 RFU, with a sharp decrease of 92% compared with the previous stage, indicating that the protein binding sites on the surface of the magnetic beads were close to saturation.

[0038] In order to verify its specificity, β-lactoglobulin and lactoferrin, α-casein, α-lactalbumin and other standard milk protein samples were selected as detection objects, and the results are shown in Figure 4As shown in Figure A, the fluorescence intensity of the β-lactoglobulin group was significantly higher than that of other protein groups (P<0.01), exceeding that of the lactoferrin, α-casein, and α-lactalbumin groups by 6.2, 6.8, and 7.1 times, respectively. This difference stems from the specific binding of β-lactoglobulin to the fluorescently labeled aptamer, which reduces the number of free fluorescent groups in solution, resulting in a significant decrease in the system's fluorescence intensity. The experimental results demonstrate that the differential fluorescence detection strategy based on nucleic acid aptamers can accurately distinguish β-lactoglobulin from other homologous proteins, validating the method's specific recognition ability for the target protein.

[0039] By linearly fitting the difference between milk adulteration rate and fluorescence intensity ( Figure 4 B) The results showed that the two exhibited a good linear correlation, with a coefficient of determination (R²) of fit. 2 The linear response rate reached 0.9318, indicating that the method has stable linear response characteristics within the experimentally set concentration range, which can meet the requirements for detecting the adulteration amount in bovine milk. Calculations showed that the limit of detection (LOD) = 3σ / sensitivity, and the reliable limit of detection for this detection system was 0.665% (based on sheep milk matrix). The linear fit corresponded to a bovine milk adulteration amount detection range of 10%–100%, effectively covering the concentration range commonly encountered in actual adulteration detection.

[0040] The preferred embodiments of the present invention have been described above, but are not intended to limit the invention. Those skilled in the art can make modifications and variations to the embodiments disclosed herein without departing from the scope and spirit of the invention.

Claims

1. A method for fluorescently labeled sheep milk quantitative detection of cow milk adulteration, comprising the following steps: (1) mixing and incubating the sample to be tested with immunomagnetic beads to obtain a suspension, removing unbound antibodies by magnetic separation, and blocking non-specific binding sites on the magnetic beads with BSA solution; the immunomagnetic beads are magnetic microspheres modified with anti-β-lactoglobulin antibodies; (2) ice-bath renaturation after thermal denaturation of 5-FAM-labeled nucleic acid aptamer BLG-14; the sequence of the 5-FAM-labeled nucleic acid aptamer BLG-14 is: FAM-5'-CGACGATCGGACCGCAGTACCCACCCACCAGCCCCAACATCATGCCCATCCGTGTGTG-3' (3) mixing and incubating the aptamer BLG-14 of step (2) with the immunomagnetic beads bound with β-lactoglobulin obtained in step (1); (4) after centrifugation, taking the supernatant, and detecting the fluorescence intensity using a fluorescence spectrophotometer or a smartphone fluorescence module.

2. The detection method according to claim 1, characterized in that The particle size of the immunomagnetic beads is 0.1-0.2 μm.

3. The method of claim 1, wherein The ratio of the amount of the anti-β-lactoglobulin antibody to the magnetic beads is 3-5:

1.

4. The method of claim 1, wherein The immunomagnetic beads are prepared by EDC / NHS chemical coupling method, in which the bovine β-lactoglobulin antibody is covalently fixed on the surface of the aminated magnetic microspheres.

5. The method of claim 1, wherein The immunomagnetic beads are blocked on the surface of the magnetic beads using 2% BSA solution.

6. The method of claim 1, wherein After mixing and incubating the sample to be tested with the immunomagnetic beads to obtain a suspension, the suspension is washed three times using PBST buffer containing 0.05% Tween-20.

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