A sample diluent and uses thereof, kits

Abstract

The application belongs to the technical field of food safety detection, and discloses a sample diluent and application and reagent kit thereof, which contain 0.5-3 g / L of alginic acid and 0.2-3 g / L of beta-cyclodextrin. The alginic acid is an anionic polyelectrolyte, has the characteristics of thickening, suspension and adsorption, the beta-cyclodextrin is a ring formed by 7 glucose residues combined by beta-1,4-glycosidic bonds, has strong coating capacity, and the two together can further reduce the influence of impurities in the sample on the detection result and inhibit the influence of the unavoidable matrix effect in the detection process.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food safety detection, and in particular to a sample diluent and application and kit thereof. BACKGROUND

[0002] The immunofluorescence chromatography technology is a detection method combining immunology and fluorescence analysis technology. Its principle is to use fluorescent microsphere-labeled antibodies or antigens to bind with the analyte, separate and enrich these complexes through chromatography, and finally measure the intensity of the fluorescence signal through an immunofluorescence analyzer to quantitatively analyze the concentration of the analyte. Its characteristics are rapidity, easy operation, convenience, high sensitivity, good accuracy, and strong specificity.

[0003] However, in the immunofluorescence chromatography detection, there is generally a matrix effect, which can cause deviation of the detection results, thereby affecting the accuracy of the detection results. The matrix is a component in the sample other than the analyte, which can significantly interfere with the analysis process of the analyte and affect the accuracy of the quantitative analysis results, causing deviation of the detection results. These influences and interferences are called matrix effects.

[0004] In the detection of mycotoxins in food safety, there are many types of feed. In order to solve the matrix effect, the sample pretreatment method and the sample diluent formula are usually used. However, the sample pretreatment method is complex and tedious, so the sample diluent is one of the methods to solve the matrix effect.

[0005] For example, CN 116879538 discloses a sample diluent and enzyme-labeled diluent for ELISA detection. The sample diluent provided in the scheme can effectively reduce the matrix interference in complex background serum and effectively maintain the uniformity and stability of the sample. The main components of the sample diluent include casein, PEG6000, sodium alginate, and BSA, and it is mainly applied to ELISA detection.

[0006] CN114252595 discloses a magnetic bead diluent for reducing sample matrix interference and an immunological detection kit. It discloses a sample diluent that can reduce sample matrix interference. The scheme adds a large amount of salt to increase the ionic strength of the diluent to reduce the binding capacity of the antibody and the protein, so that the heteroprotein in the blood plasma cannot interact with the antibody through weak interaction, reducing the possible non-specific antigen-antibody binding, thereby reducing the influence of the sample matrix effect.

[0007] The problem to be solved by the present application is how to provide a sample diluent that is different from the prior art and can inhibit the matrix effect. SUMMARY

[0008] The sample diluent can slow down the flow rate of the system in the chromatography process and adsorb impurities in the sample, thereby reducing the influence of the matrix effect on the detection result through the addition of alginic acid and beta-cyclodextrin.

[0009] To achieve the above-mentioned object, the application provides a sample diluent, which comprises alginic acid with a concentration of 0.5-3 g / L and beta-cyclodextrin with a concentration of 0.2-3 g / L.

[0010] Preferably, the sample diluent specifically comprises HEPES with a concentration of 1-3 g / L, surfactant S9 with a concentration of 3-10 g / L, BSA with a concentration of 3-10 g / L, trehalose with a concentration of 5-20 g / L, alginic acid with a concentration of 0.5-3 g / L, beta-cyclodextrin with a concentration of 0.2-3 g / L and Proclin-300 with a concentration of 0.2-1 g / L, and the solvent is deionized water.

[0011] Preferably, the sample diluent specifically comprises HEPES with a concentration of 1-3 g / L, surfactant S9 with a concentration of 3-8 g / L, BSA with a concentration of 3-8 g / L, trehalose with a concentration of 5-15 g / L, alginic acid with a concentration of 0.5-2 g / L, beta-cyclodextrin with a concentration of 0.2-1 g / L and Proclin-300 with a concentration of 0.2-1 g / L, and the solvent is deionized water.

[0012] In addition, the application also discloses the use of the sample diluent as described above for diluting a sample in an immunofluorescence chromatography test of food.

[0013] Preferably, the food is feed.

[0014] Preferably, the immunofluorescence chromatography test is a test for vomitoxin, aflatoxin or zearalenone.

[0015] Preferably, the aflatoxin is specifically aflatoxin B1.

[0016] In addition, the application also discloses the use of the sample diluent as described above for preparing an ELISA detection kit.

[0017] The application has the following beneficial effects:

[0018] In the sample diluent of the application, alginic acid is an anionic polyelectrolyte, which has the characteristics of thickening, suspension aiding and adsorption. In the chromatography process, the thickening effect can adjust the flow rate of the system; the suspension aiding effect can prevent the aggregation and precipitation of feed sample particles, so that the sample can maintain a uniform dispersed state in the chromatography system; and the adsorption effect can adsorb some components of the sample, thereby achieving the separation effect.

[0019] Beta-cyclodextrin is a ring composed of 7 glucose residues combined by beta-1, 4-glycosidic bond, which has strong inclusion capacity and can form inclusion complexes with various compounds. It has certain recognition, improves the stability and solubility of the sample. Beta-cyclodextrin can form a complex with alginic acid, thereby improving the accuracy of the analysis result and playing an important role in the chromatography process.

[0020] The two together can further reduce the influence of impurities in the sample on the detection result and inhibit the influence of the unavoidable matrix effect in the detection process. DETAILED DESCRIPTION

[0021] The present application will be described clearly and completely in combination with the embodiments of the present application. In the description of the present application, it should be noted that the specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased on the market.

[0022] The source information of the raw materials involved in the present application and used in the detection process is shown in Tables 1-2:

[0023] Table 1: Raw material information table

[0024] Raw materials Supplier Raw materials Supplier HEPES Zengsheng Biotech (Guangzhou) Co., Ltd. Alginic acid Zengsheng Biotech (Guangzhou) Co., Ltd. Surfactant S9 Shanghai Jingwen Technology Co., Ltd. Proclin-300 Shanghai Shengbo Biological Technology Co., Ltd. BSA Hezuo Biological Technology (Shanghai) Co., Ltd. Trehalose Kunshan Yuanmu Biological Technology Co., Ltd. β-Cyclodextrin Shanghai Baiss Biological Technology Co., Ltd. Guangzhou Branch

[0025] Table 2: Detection raw material information table

[0026]

[0027] Preparation method of sample diluent

[0028] HEPES, surfactant S9, BSA, trehalose, alginic acid, beta-cyclodextrin and Proclin-300 are dissolved in deionized water according to the predetermined concentration to obtain a sample diluent. More specifically, the specific amount of each raw material in the sample diluent is shown in the specific description of each embodiment and comparative example.

[0029] Example 1

[0030] A sample diluent, with deionized water as the solvent, further comprises the following components (solute):

[0031] 1g / L of HEPES;

[0032] 10g / L of surfactant S9;

[0033] 3g / L of BSA;

[0034] 20g / L of trehalose;

[0035] 0.5g / L of alginic acid;

[0036] 3 g / L of β-cyclodextrin;

[0037] 0.2 g / L of Proclin-300.

[0038] Example 2

[0039] A sample diluent, with deionized water as solvent, and further comprising the following components (solute):

[0040] 3 g / L of HEPES;

[0041] 3 g / L of surfactant S9;

[0042] 10 g / L of BSA;

[0043] 5 g / L of trehalose;

[0044] 3 g / L of alginic acid;

[0045] 0.2 g / L of β-cyclodextrin;

[0046] 1 g / L of Proclin-300.

[0047] Example 3

[0048] A sample diluent, with deionized water as solvent, and further comprising the following components (solute):

[0049] 1 g / L of HEPES;

[0050] 8 g / L of surfactant S9;

[0051] 3 g / L of BSA;

[0052] 15 g / L of trehalose;

[0053] 0.5 g / L of alginic acid;

[0054] 1 g / L of β-cyclodextrin;

[0055] 0.2 g / L of Proclin-300.

[0056] Example 4

[0057] A sample diluent, with deionized water as solvent, and further comprising the following components (solute):

[0058] 3 g / L of HEPES;

[0059] 3 g / L of surfactant S9;

[0060] 8 g / L of BSA;

[0061] 5 g / L of trehalose;

[0062] 2 g / L of alginic acid;

[0063] 0.2 g / L of β-cyclodextrin;

[0064] 1 g / L of Proclin-300.

[0065] Example 5

[0066] A sample diluent, with deionized water as solvent, and further comprising the following components (solute):

[0067] 2 g / L of HEPES;

[0068] 5 g / L of surfactant S9;

[0069] 5 g / L of BSA;

[0070] 10 g / L of trehalose;

[0071] 1 g / L of alginic acid;

[0072] 0.5 g / L of β-cyclodextrin;

[0073] 0.5 g / L of Proclin-300.

[0074] Comparative Example 1

[0075] A diluent, with deionized water as solvent, and further comprising the following components (solute):

[0076] 2 g / L of HEPES;

[0077] 5 g / L of surfactant S9;

[0078] 5 g / L of BSA;

[0079] 10 g / L of trehalose;

[0080] 1.5 g / L of β-cyclodextrin;

[0081] 0.5 g / L of Proclin-300.

[0082] Comparative Example 2

[0083] A diluent, with deionized water as solvent, and further comprising the following components (solute):

[0084] 2 g / L of HEPES;

[0085] 5 g / L of surfactant S9;

[0086] 5 g / L of BSA;

[0087] 10 g / L of trehalose;

[0088] 1.5 g / L of alginic acid;

[0089] 0.5 g / L of Proclin-300.

[0090] Performance verification experiment

[0091] First part: suitability test of vomitoxin

[0092] The sample diluent prepared by each of the above examples and comparative examples was used to test the content of vomitoxin in liquid standard 1 (Romer Labs) and solid quality control (Romer Labs, National Food and Material Reserve Bureau Scientific Research Institute), and the consistency of the test value with the labeled value was compared.

[0093] The original concentration of liquid standard 1 was 100 μg / mL, and was diluted to a concentration of 5000 ppb, 2500 ppb, 1250 ppb, 625 ppb, 312 ppb, 156 ppb, and 78 ppb.

[0094] The test results are shown in Tables 3-4:

[0095] Table 3: Comparison table of labeled concentration and test value of examples 1-4

[0096]

[0097] Table 4: Comparison table of labeled concentration and test value of examples 5 and comparative examples 1-2

[0098]

[0099] Second part: suitability test of aflatoxin B1

[0100] The sample diluent prepared by each of the above examples and comparative examples was used to test the content of vomitoxin in liquid standard (Romer Labs) and solid quality control (Romer Labs, National Food and Material Reserve Bureau Scientific Research Institute), and the consistency of the test value with the labeled value was compared, and the test results are shown in Tables 5 and 6.

[0101] The original concentration of liquid standard 2 was 2 μg / mL, and was diluted to a concentration of 75 ppb, 37.5 ppb, 18.75 ppb, 9.38 ppb, 4.69 ppb, 2.34 ppb, and 1.17 ppb.

[0102] Table 5: Comparison table of labeled concentration and test value of examples 1-4

[0103]

[0104] Table 6: Comparison table of labeled concentration and test value of Example 5 and Comparative Examples 1-2

[0105]

[0106] Part III: Applicability test of zearalenone

[0107] The sample diluent prepared by each of the above examples and comparative examples was used to test the liquid standard (Romer Labs) and solid quality control (Romer Labs, National Food and Material Reserve Bureau Scientific Research Institute) for zearalenone content, and the compliance of the labeled value and the test value. The test results are shown in Tables 7 and 8.

[0108] The original concentration of the liquid standard 3 was 100㎍ / mL, and was diluted to concentrations of 1000ppb, 500ppb, 125ppb, 62.5ppb, 31.25ppb, 15.63ppb, and 7.81ppb.

[0109] Table 7: Comparison table of labeled concentration and test value of Examples 1-4

[0110]

[0111] Table 8: Comparison table of labeled concentration and test value of Example 5 and Comparative Examples 1-2

[0112]

[0113] Result analysis

[0114] 1. According to Tables 3-4, it can be seen from Examples 1-5 that, during the test of vomitoxin, Examples 3-5 have more stable recovery rates (80-120%) than Examples 1-2, which means that the sample diluent formula disclosed in Examples 3-5 has more advantages;

[0115] Further observation of Comparative Examples 1-2 shows that, when either β-cyclodextrin or alginic acid is missing from the sample diluent, the stability of the recovery rate of Comparative Examples 1-2 has a downward trend to varying degrees compared to Examples 1-5, which means that the absence of either β-cyclodextrin or alginic acid in the sample diluent has a relatively obvious impact on the stability of the recovery rate during the test. The decrease in the stability of the recovery rate also means a decrease in the detection accuracy. It is speculated that the reason for this phenomenon is that β-cyclodextrin (β-CD) can form a complex with alginic acid. β-cyclodextrin is a cyclic oligosaccharide with a truncated cone-shaped cavity structure. Alginic acid is a natural linear polysaccharide formed by alternating β-D-mannuronic acid (M) and α-L-guluronic acid (G) through 1,4-glycosidic bonds.

[0116] The complexation of β-CD and alginate is mainly realized by "hydrophobic cavity inclusion" and "hydrogen bonding", supplemented by electrostatic and conformational regulation. The hydrophobic region of alginate can be included in the cavity of β-CD to form a "host-guest complex". This inclusion shields the hydrophobic group, reduces non-specific adsorption, and improves the selectivity of chromatographic separation. The hydroxyl groups on the outer wall of β-CD form hydrogen bonds with the carboxyl groups or hydroxyl groups on the sugar ring of alginate, enhancing the stability of their combination. After the combination of β-CD and alginate, the chain conformation or aggregation state of alginate can be changed, reducing its non-specific adsorption, thereby reducing background interference in chromatography and improving the separation efficiency of target substances.

[0117] Therefore, the interaction between β-CD and alginate can effectively reduce interference and enhance separation selectivity in chromatography, thereby improving analysis accuracy.

[0118] 2. Further observation of Tables 5-8 shows that in the detection process of aflatoxin B1 and zearalenone, there is still a certain trend in Examples 3-5 compared to Examples 1-2, but the recovery stability of Examples 1-4 has decreased. It can be seen that the sample diluent prepared in the present application is more suitable for the test of vomitoxin.

[0119] At the same time, it is worth noting that the sample diluent prepared in Example 5 has shown good detection accuracy in the test of multiple toxins.

Claims

1. A sample diluent, characterized in that, The sample diluent comprises HEPES at a concentration of 1-3 g / L, S9 at 3-8 g / L, BSA at 3-8 g / L, trehalose at 5-15 g / L, alginic acid at 0.5-2 g / L, β-cyclodextrin at 0.2-1 g / L and Proclin-300 at 0.2-1 g / L, and the solvent is deionized water.

2. Use of the sample diluent according to claim 1 for diluting samples and inhibiting matrix effects in immunofluorescence chromatographic tests of food.

3. Use according to claim 2, characterized in that, The food is feed.

4. Use according to claim 2, characterized in that, The immunofluorescence chromatographic test is a test for vomitoxin, aflatoxin or zearalenone.

5. Use according to claim 4, characterized in that, The aflatoxin is aflatoxin B1.

6. Use of the sample diluent according to claim 1 for preparing an ELISA detection kit.

7. An ELISA test kit characterized in that, containing the sample diluent according to claim 1.

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