Method for simultaneously detecting multiple allergens in food

Abstract

The invention relates to a method for simultaneously detecting multiple allergens in food, which comprises the following steps: diluting multiple target allergen proteins according to a concentration gradient, adding into an elisa plate, and incubating overnight; washing the elisa plate by using PBST washing liquor to remove uncombined proteins; adding 5% of BSA confining liquid into the elisa plate, oscillating and incubating, and washing off the confining liquid; the method comprises the following steps: premixing aptamers of a plurality of target allergen proteins into a premixed solution; adding the premixed solution into the elisa plate from which the confining liquid is washed off, oscillating and incubating, and washing off the premixed solution; adding ultrapure water into the elisa plate, incubating at 95 DEG C to denature the allergen protein, and recovering supernate containing the aptamer; taking the supernatant containing the aptamer as a DNA template, and carrying out Taqman qPCR amplification and fluorescence signal detection to obtain a reaction ct value; and drawing a standard curve by taking the reaction ct value as a vertical coordinate and the target allergen protein concentration as a horizontal coordinate. The method can be used for simultaneously detecting the content of various allergic proteins in food, and is high in efficiency and strong in specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of food allergen detection, and in particular to a method for simultaneously detecting multiple allergens in food. Background Art

[0002] The prevalence of food allergies in adults is about 2%, while in children it is as high as 8%. Since lower concentrations of allergens can also cause severe immune reactions; the main means of treating food allergies is still to avoid contact with allergens. Therefore, it is crucial to establish more sensitive and accurate food allergen detection technology. In 2013, the World Health Organization (WHO) listed milk, eggs, soybeans, peanuts, wheat, nuts, fish, and crustacean alpha animals as the "eight major allergens in the world", among which gliadin and Ara h1 protein are the main proteins that cause allergic immune reactions.

[0003] There are many methods for detecting food allergens, mainly immunological methods, such as enzyme-linked immunosorbent assay (ELISA), and molecular biology methods, such as real-time fluorescence quantitative PCR. These methods have their own advantages and disadvantages. Although ELISA has a short detection time, the stability of biological antibodies during the detection process is not good. Although real-time fluorescence quantitative PCR can effectively overcome the influence of changes in the protein structure of allergens, it can only target the gene sequence encoding the target protein, and the instrument operation is relatively complicated and expensive. Therefore, it is necessary to develop a new method for detecting food allergens. Summary of the invention

[0004] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the object of the present invention is to provide a method for simultaneously detecting multiple allergens in food.

[0005] To this end, the present invention proposes a method for simultaneously detecting multiple allergens in food, which comprises the following steps:

[0006] (1) Dilute multiple target allergen proteins according to a concentration gradient, add them to the ELISA plate, and incubate overnight;

[0007] (2) Wash the ELISA plate with PBST to remove unbound proteins;

[0008] (3) Add 5% BSA blocking solution to the ELISA plate, shake and incubate, then wash away the blocking solution;

[0009] (4) premixing a plurality of aptamers of the target allergen protein into a premix solution;

[0010] (5) adding the premixed solution to the ELISA plate from which the BSA blocking solution has been washed off in step (3), shaking and incubating, and then washing off the premixed solution;

[0011] (6) adding ultrapure water to the ELISA plate, incubating at 95° C. to denature the allergen protein, and recovering the supernatant containing the aptamer;

[0012] (7) using the supernatant containing the aptamer as a DNA template, performing Taqman qPCR amplification and fluorescence signal detection to obtain a reaction ct value;

[0013] (8) Draw a standard curve with the reaction ct value as the ordinate and the target allergen protein concentration as the abscissa.

[0014] According to the method for simultaneously detecting multiple allergens in food of the present invention, the method utilizes aptamers to specifically identify target allergen proteins. When the allergen proteins denature and change their tertiary structure, the aptamers cannot bind to them. The content of the target allergen proteins is indirectly reflected by measuring the content of the aptamers in the system. In this way, the target genomic DNA in the traditional qPCR method for detecting allergen proteins is replaced by aptamer molecules, and the complex and time-consuming target DNA extraction process is eliminated. The method has good sensitivity, can simultaneously detect the contents of multiple allergen proteins in food, has high efficiency and strong specificity, and can play an important role in food allergen detection and other aspects.

[0015] Optionally, the target allergen protein includes gliadin and peanut Ara h1 protein; the nucleotide sequence of the aptamer of gliadin is shown in SEQ ID NO:1; the nucleotide sequence of the aptamer of peanut Ara h1 protein is shown in SEQ ID NO:2.

[0016] Furthermore, in Taqman qPCR detection, the 5' end of the Taqman probe used for the gliadin protein was modified by FAM, and the 3' end was labeled with a BHQ1 group; the 5' end of the Taqman probe used for the peanut Ara h1 protein was modified by VIC, and the 3' end was labeled with a BHQ1 group.

[0017] Furthermore, in Taqman qPCR detection, the primers and probe sequences used for the gliadin protein are shown in SEQ ID NOs: 3 to 5; the primers and probe sequences used for the peanut Ara h1 protein are shown in SEQ ID NOs: 6 to 8.

[0018] Optionally, the standard curve is drawn to satisfy the relationship R 2 ≥0.98.

[0019] Optionally, the reaction system of Taqman qPCR amplification is: total reaction volume 20 μL, Taqman Probe Master Mix 10 μL, 10 μM upstream primer 0.4 μL, 10 μM downstream primer 0.4 μL, 10 μM Taqman Probe 0.2 μL, DNA template 1 μL, sterile water 8 μL;

[0020] The reaction procedure of the Taqman qPCR amplification was: denaturation at 95°C for 1 min; denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s, while collecting the fluorescence signal, for a total of 40 cycles.

[0021] Optionally, in step (3), the blocking solution is added and incubated with shaking for 2 hours.

[0022] Optionally, in step (5), the premix is ​​added and incubated with shaking for 1 hour.

[0023] Optionally, in step (6), the mixture is incubated at 95° C. for 12 minutes.

[0024] The present invention also proposes the application of the above method for simultaneously detecting multiple allergens in food in detecting food allergens. After diluting the concentration of the protein to be detected, the reaction ct value is obtained using the method.

[0025] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a standard curve of gliadin according to an embodiment of the present invention;

[0027] Figure 2 is a standard curve of peanut Ara h1 protein according to an embodiment of the present invention;

[0028] Figure 3 The specific results of gliadin according to the embodiments of the present invention;

[0029] Figure 4 The specific results of peanut Ara h1 protein according to the embodiment of the present invention;

[0030] Figure 5 This is the detection result of β-lactoglobulin according to an embodiment of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

[0032] In order to better understand the above technical scheme, the exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0033] The test materials used in the present invention are all common commercial products and can be purchased on the market; the experiments involved are all conventional experimental methods unless otherwise specified.

[0034] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0035] Example 1

[0036] (1) The two target allergen proteins (gliadin and peanut Ara h1 protein) were diluted according to a concentration gradient (0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 12 μg / mL, 16 μg / mL, 20 μg / mL), added to the ELISA plate, 100 μL per well, and incubated overnight.

[0037] (2) Add 200 μL PBST (PBS containing 0.1% Tween-20) to each well of the ELISA plate, then pat dry. Repeat three times to remove unbound target protein.

[0038] (3) Add 5% BSA blocking solution to the ELISA plate, 200 μL per well, incubate with shaking for 2 h, wash three times with PBST solution, and pat dry.

[0039] (4) The aptamers of the two target allergen proteins are premixed into a premix solution, and the concentration of the premix solution is 20 nM. The sequences of the aptamer of gliadin and the aptamer of peanut Ara h1 protein are shown in Table 1 below.

[0040] Table 1 Aptamer sequences of gliadin and peanut Ara h1 protein

[0041]

[0042] (5) Add the premixed solution to the ELISA plate from which the blocking solution has been washed off in step (3), add 100 μL of the premixed solution to each well, incubate with shaking for 1 hour, wash away the premixed solution, and wash five times with 200 μL of PBST buffer.

[0043] (6) Add ultrapure water to the ELISA plate, add 50 μL of MilliQ water to each well; incubate the ELISA plate at 95° C. for 12 min to denature the target allergen protein. After the plate is cooled to room temperature, take 30 μL of the supernatant from each well of the ELISA plate and transfer it to a centrifuge tube for later use.

[0044] (7) Using the supernatant containing the aptamer as a DNA template, Taqman qPCR amplification and fluorescence signal detection were performed. The 5' end of the Taqman probe used for gliadin was modified by FAM and the 3' end was labeled by the BHQ1 group; the 5' end of the Taqman probe used for peanut Arah1 protein was modified by VIC and the 3' end was labeled by the BHQ1 group. In the Taqman qPCR detection, the sequences of the probes and primers used for the gliadin and peanut Arah1 protein are shown in Table 2.

[0045] Table 2 Primer and probe sequences

[0046]

[0047] The reaction system of Taqman qPCR amplification was as follows: total reaction volume 20 μL, Taqman Probe MasterMix 10 μL, 10 μM upstream primer 0.4 μL, 10 μM downstream primer 0.4 μL, 10 μM Taqman Probe 0.2 μL, DNA template 1 μL, and sterile water 8 μL.

[0048] The reaction program of Taqman qPCR amplification was as follows: denaturation at 95°C for 1 min; denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s, and collection of fluorescence signals at the same time, for a total of 40 cycles.

[0049] (7) Read the reaction ct value and draw a standard curve with ct value as the ordinate and target allergen protein concentration (μg / ml) as the abscissa. Set the abscissa to logarithmic form and use the logistic model in Origin software to perform four-parameter fitting. Then draw a standard curve, as shown in the following figure: Figure 1 and Figure 2The results showed that there was a good linear relationship between protein concentration and ct value, and the correlation coefficient R 2 The linear regression equation of the standard curve is Y = -1.20*X + 22.86. The correlation coefficient R of the peanut Ara h1 group 2 is 0.985, and the linear regression equation of the standard curve is Y=-2.38*X+20.57.

[0050] Example 2 Specificity test

[0051] The protein concentration of egg white albumin (OVA), β-lactoglobulin (β-Lg), α-casein, and a mixed group of gliadin and peanut Arah1 protein (the ratio of the two proteins was 1:1) was diluted to 10 μg / ml, and the ct value was detected using the established detection method based on aptamers and Taqman probes (Example 1). The obtained data were analyzed by independent sample t test using software GraphPad Prism 9, and significant differences were indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0052] The results are as follows Figure 3 and Figure 4 As shown, this method has good specificity for gliadin and peanut Ara h1 protein.

[0053] Example 3 Determination of Precision

[0054] The precision of this application is affected by many factors, such as the number of plate washes, the choice of reagents, etc. The precision is determined by evaluating the intra-batch and inter-batch differences.

[0055] (1) Within the batch: Gliadin and peanut Ara h1 protein were mixed in a ratio of 1:1 and diluted to three gradient concentrations of 5 μg / mL, 10 μg / mL and 20 μg / mL. Five replicates were made for each concentration. The ct value was detected using the established detection method based on aptamer and Taqman probe (Example 1), and the coefficient of variation (CV) was calculated.

[0056] (2) Between batches: Gliadin and peanut Ara h1 protein were mixed in a ratio of 1:1 and diluted to three gradient concentrations of 5 μg / mL, 10 μg / mL and 20 μg / mL. The ct value was detected using the established aptamer and Taqman probe-based detection method (Example 1). Each concentration was measured once a day for 5 consecutive days.

[0057] The differences within and between batches can be expressed by the coefficient of variation (CV), CV = SD / X*100%, where SD is the standard deviation and X is the mean. Usually CV ≤ 15%, indicating that the method has good precision.

[0058] The results are shown in Tables 3 and 4. The coefficients of variation are all greater than 15%, which has good accuracy.

[0059] Table 3 Precision results of gliadin

[0060]

[0061]

[0062] Table 4 Precision results of peanut Ara h1 protein

[0063]

[0064] Example 4 Detection of β-lactoglobulin

[0065] (1) β-lactoglobulin was diluted according to a concentration gradient (0.1 μg / mL, 1 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL), added to an ELISA plate, 100 μL per well, and incubated overnight.

[0066] (2) Add 200 μL PBST (PBS containing 0.1% Tween-20) to each well of the ELISA plate, then pat dry. Repeat three times to remove unbound target protein.

[0067] (3) Add 5% BSA blocking solution to the ELISA plate, 200 μL per well, incubate with shaking for 2 h, wash three times with PBST solution, and pat dry.

[0068] (4) The above aptamers of β-lactoglobulin were premixed into premix solutions, and the concentration of the premix solutions was 20 nM. The sequences of the aptamers of β-lactoglobulin are shown in Table 5 below.

[0069] Table 5 Aptamer sequences of β-lactoglobulin

[0070]

[0071] (5) Add the premixed solution to the ELISA plate from which the blocking solution has been washed off in step (3), add 100 μL of the premixed solution to each well, incubate with shaking for 1 hour, wash away the premixed solution, and wash five times with 200 μL of PBST buffer.

[0072] (6) Add ultrapure water to the ELISA plate, add 50 μL of MilliQ water to each well; incubate the ELISA plate at 95° C. for 12 min to denature the target allergen protein. After the plate is cooled to room temperature, take 30 μL of the supernatant from each well of the ELISA plate and transfer it to a centrifuge tube for later use.

[0073] (7) Using the supernatant containing the aptamer as a DNA template, qPCR amplification and fluorescence signal detection were performed. In the qPCR detection, the sequences of the probe and primers used for the β-lactoglobulin were shown in Table 6.

[0074] Table 6 β-lactoglobulin probe and primer sequences

[0075]

[0076] The reaction system of Taqman qPCR amplification was as follows: total reaction volume 20 μL, Taqman Probe MasterMix 10 μL, 10 μM upstream primer 0.4 μL, 10 μM downstream primer 0.4 μL, 10 μM Taqman Probe 0.2 μL, DNA template 1 μL, and sterile water 8 μL.

[0077] The reaction program of Taqman qPCR amplification was as follows: denaturation at 95°C for 1 min; denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s, and collection of fluorescence signals at the same time, for a total of 40 cycles.

[0078] (7) Read the reaction ct value and compare it with the uncoated protein group.

[0079] The results are as follows Figure 5 As shown, the ct value of the protein-coated group was significantly lower than that of the uncoated group.

[0080] In summary, according to the embodiments of the present invention, aptamers can be used to specifically identify target allergen proteins. When the allergen protein denatures and changes its tertiary structure, the aptamer cannot bind to it. The content of the target allergen protein is indirectly reflected by measuring the content of the aptamer in the system. In this embodiment, specific probes are designed for the gliadin aptamer (G33) and the peanut Ara h1 protein aptamer (A1), and the experimental conditions are determined, thereby establishing a method for simultaneously detecting two allergens (gliadin aptamer and peanut Ara h1 protein) in food. In this way, the target genomic DNA in the traditional qPCR method for detecting allergen proteins is replaced by aptamer molecules, and the complex and time-consuming target DNA extraction process is eliminated. The method has good sensitivity, can simultaneously detect the content of multiple allergen proteins in food, has high efficiency and strong specificity, and can play an important role in food allergen detection and the like.

[0081] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0082] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for simultaneously detecting multiple allergens in food, characterized in that: The following steps are involved: (1) Dilute multiple target allergen proteins according to a concentration gradient, add them to the ELISA plate, and incubate overnight; (2) Wash the ELISA plate with PBST to remove unbound proteins; (3) Add 5% BSA blocking solution to the ELISA plate, shake and incubate, then wash away the blocking solution; (4) premixing a plurality of aptamers of the target allergen proteins into a premix solution; (5) adding the premixed solution to the ELISA plate from which the blocking solution has been washed off in step (3), shaking and incubating, and then washing off the premixed solution; (6) adding ultrapure water to the ELISA plate, incubating at 95° C. to denature the allergen protein, and recovering the supernatant containing the aptamer; (7) using the supernatant containing the aptamer as a DNA template, performing Taqman qPCR amplification and fluorescence signal detection to obtain a reaction ct value; (8) Draw a standard curve with the reaction ct value as the ordinate and the target allergen protein concentration as the abscissa.

2. The method for simultaneously detecting multiple allergens in food according to claim 1, characterized in that: The target allergen proteins include gliadin and peanut Ara h1 protein; the nucleotide sequence of the aptamer for gliadin is shown in SEQ ID NO: 1; the nucleotide sequence of the aptamer for peanut Ara h1 protein is shown in SEQ ID NO:

2.

3. The method for simultaneously detecting multiple allergens in food according to claim 2, characterized in that: In Taqman qPCR detection, the 5' end of the Taqman probe used for the gliadin protein was modified by FAM, and the 3' end was labeled with a BHQ1 group; the 5' end of the Taqman probe used for the peanut Ara h1 protein was modified by VIC, and the 3' end was labeled with a BHQ1 group.

4. The method for simultaneously detecting multiple allergens in food according to claim 3, characterized in that: In Taqman qPCR detection, the sequences of the primers and probes used for the gliadin protein are shown in SEQ ID NOs: 3 to 5; the sequences of the primers and probes used for the peanut Ara h1 protein are shown in SEQ ID NOs: 6 to 8.

5. The method for simultaneously detecting multiple allergens in food according to claim 1, characterized in that: In step (8), the standard curve drawn satisfies the relationship number R 2 ≥0.

98.

6. The method for simultaneously detecting multiple allergens in food according to any one of claims 1 to 5, characterized in that: The reaction system of the Taqman qPCR amplification is as follows: the total reaction volume is 20 μL, Taqman Probe Master Mix is ​​10 μL, 10 μM upstream primer is 0.4 μL, 10 μM downstream primer is 0.4 μL, 10 μM Taqman Probe is 0.2 μL, DNA template is 1 μL, and sterile water is 8 μL; The reaction procedure of the Taqman qPCR amplification was: denaturation at 95°C for 1 min; denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s, while collecting the fluorescence signal, for a total of 40 cycles.

7. The method for simultaneously detecting multiple allergens in food according to claim 1, characterized in that: In the step (3), the blocking solution is added and incubated with shaking for 2 hours.

8. The method for simultaneously detecting multiple allergens in food according to claim 1, characterized in that: In the step (5), the premixed solution is added and incubated with shaking for 1 hour.

9. The method for simultaneously detecting multiple allergens in food according to claim 1, characterized in that: In the step (6), the mixture is incubated at 95° C. for 12 minutes.

10. Use of the method for simultaneously detecting multiple allergens in food according to any one of claims 1 to 9 in detecting food allergens, characterized in that: After diluting the concentration of the protein to be tested, the reaction ct value was obtained using the method described above.

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