Fluorescence immunodetection card for simultaneously detecting various mycotoxins in cereals and application of fluorescence immunodetection card

Abstract

The invention belongs to the technical field of agricultural science, and particularly relates to a three-in-one fluorescent quantitative rapid detection card for simultaneously detecting zearalenone, aflatoxin B1 and vomitoxin in grains and application of the three-in-one fluorescent quantitative rapid detection card. The rapid detection card is accurate and reliable in identification result and high in sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of agricultural science and technology, and specifically relates to a three-in-one fluorescent quantitative detection card for simultaneously detecting zearalenone, aflatoxin B1 and vomitoxin in grains and an application thereof. Background Art

[0002] Mycotoxins are toxic metabolites secreted by fungi. They are widely found in agricultural products such as grains, oils, fruits and nuts, resulting in reduced yields and lower quality of agricultural products. Mycotoxin contamination is difficult to predict and avoid, and can occur during the planting, harvesting, storage and drying of crops. Due to their good stability to chemical, physical and thermal treatments, many mycotoxins are extremely difficult to eliminate during food production and processing. When mycotoxins enter the human and animal bodies through food, they can cause adverse health effects in the body and even lead to death.

[0003] Since mycotoxins are widely present in cereals and their products worldwide and show toxicity at low dose levels, various rapid and sensitive mycotoxin detection methods are being developed. Current detection methods include thin layer chromatography (TLC), instrumental analysis, immunoassay, electrochemical detection, surface plasmon resonance technology (SPR) and biochip technology, among which instrumental analysis and immunoassay are the most common. Instrumental analysis includes high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UPLC) and liquid chromatography tandem mass spectrometry (LC-MS / MS). Immunoassay includes enzyme-linked immunosorbent assay (ELISA), immunoaffinity chromatography (IAC), chemiluminescent enzyme immunoassay (CLEIA), colloidal gold immunochromatography (GICA), fluorescence polarization immunoassay (FPIA), etc. Instrumental analysis has high sensitivity, high accuracy and good repeatability, but the equipment is expensive, the sample preparation is complicated, and professional technicians are required to operate, which is not suitable for rapid detection of large-scale samples. The immunoassay method uses antibodies as core recognition elements for detection. It is fast, low-cost, and easy to operate, and can achieve high-throughput on-site screening of a large number of samples. Summary of the invention

[0004] In view of the problems and current status of the prior art, the present invention has developed a three-in-one fluorescent quantitative rapid test card for zearalenone / aflatoxin B1 / vomitoxin based on time-resolved fluorescent nano-microspheres (abbreviated as ZAD fluorescent quantitative rapid test card), which can be used for indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and fluorescent immunochromatographic test strips for rapid detection of mycotoxins in actual samples. The technical performance of the three-in-one fluorescent quantitative rapid test card for mycotoxins was verified by testing a variety of grain and feed samples, realizing the all-in-one rapid, simple, accurate, quantitative and automated detection of mycotoxins in grains.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a fluorescent immunoassay card, comprising a sample pad, a marking pad, a nitrocellulose membrane and an absorption pad arranged in sequence, wherein the marking pad is sprayed with a monoclonal antibody solution labeled with fluorescent microspheres, and the monoclonal antibody solution is a zearalenone-specific monoclonal antibody, aflatoxin-specific monoclonal antibody and vomitoxin-specific monoclonal antibody.

[0007] According to the present invention, the concentration of ZEN (zearalenone-specific monoclonal antibody) is 4-7 mg / mL, preferably 5-6 mg / mL, preferably 5.6 mg / mL; the concentration of AFB1 (aflatoxin-specific monoclonal antibody) is 3-6 mg / mL, preferably 4-5 mg / mL, preferably 4.4 mg / mL; the concentration of DON (deoxyribonucleic acid-specific monoclonal antibody) is 3-6 mg / mL, preferably 4-5 mg / mL, preferably 4.9 mg / mL.

[0008] According to the present invention, a detection line and a quality control line are sequentially arranged in parallel on the nitrocellulose membrane, and the detection line is coated with a corresponding protein that undergoes an antigen-antibody reaction with the sample to be tested.

[0009] According to the present invention, the detection line is coated with zearalenone-BSA, aflatoxin B1-BSA, and vomitoxin-BSA.

[0010] According to the present invention, the concentration of zearalenone-BSA is 2-5 mg / mL, preferably 3-4 mg / mL, preferably 3.3 mg / mL; the concentration of aflatoxin B1-BSA is 2-5 mg / mL, preferably 3-4 mg / mL, preferably 3.3 mg / mL; the concentration of vomitoxin-BSA is 1-4 mg / mL, preferably 2-3 mg / mL, preferably 2.6 mg / mL.

[0011] According to the present invention, the quality control line is coated with a corresponding antigen or antibody that undergoes an antigen-antibody binding reaction with the labeled antibody or antigen.

[0012] According to the present invention, the quality control line is coated with anti-mouse IgG antibody, anti-rabbit IgG antibody or anti-chicken IgG antibody, preferably anti-mouse IgG antibody, and the quality control line is coated with goat anti-mouse secondary antibody.

[0013] According to the present invention, the width of the sample pad and the absorption pad is about 20-30 mm.

[0014] According to the present invention, the width of the marking pad is about 8-12 mm.

[0015] According to the present invention, the width of the fluorescent immunoassay card is 2-5 mm.

[0016] In a second aspect, the present invention provides a detection kit, comprising the fluorescent immunoassay card as described in the first aspect.

[0017] In a third aspect, the present invention provides a detection method for a test strip, using the test strip as described in the first aspect and the kit as described in the second aspect, the detection method comprising the following steps: adding a sample to be tested to a spotting well, reacting with immunochromatography for 5-10 minutes, and observing color changes.

[0018] In a fourth aspect, the present invention provides use of the fluorescent immunoassay card described in the first aspect in detecting multiple fungal toxins in cereals.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention simultaneously labels zearalenone, aflatoxin B1 and vomitoxin with fluorescent microspheres to develop a three-way test card that can simultaneously detect zearalenone, aflatoxin B1 and vomitoxin;

[0021] (2) The three-in-one quantitative mycotoxin detection card provided by the present invention can specifically detect zearalenone, aflatoxin B1 and vomitoxin in different grains, and the identification results are accurate, reliable and highly sensitive;

[0022] (3) The three-in-one fluorescent quantitative rapid test card for zearalenone / aflatoxin B1 / deoxynivalenol provided by the present invention has a detection sensitivity of 150 ppb for zearalenone, 20 ppb for aflatoxin B1, and 500 ppb for deoxynivalenol, which are significantly higher than the national standard. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / domitoxin. The line can be eliminated when the concentration of zearalenone ZEN is 150ppb.

[0024] Figure 2 It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / domitoxin, which can eliminate the line when the concentration of aflatoxin B1 is 20ppb.

[0025] Figure 3 It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / deoxynivalenol. It can eliminate the line when the concentration of deoxynivalenol DON is 500ppb.

[0026] Figure 4 It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / deoxynivalenol, and shows the color development of aflatoxin B1 at different added concentrations.

[0027] Figure 5 Read using a fluorescent immunoassay analyzer Figure 4 Detect the values ​​of the T line and C line of the test card, calculate the T / C ratio, and draw a graph of T / C and aflatoxin B1 concentration.

[0028] Figure 6 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for aflatoxin B1 concentration of 0, with a CV value of 4.6%.

[0029] Figure 7 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for aflatoxin B1 concentration of 0.05ppb and the CV value is 4.5%.

[0030] Figure 8 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for aflatoxin B1 concentration of 0.5ppb and the CV value is 3.3%.

[0031] Fig. 9 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for aflatoxin B1 concentration of 1ppb with a CV value of 4.9%.

[0032] Fig.10 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for aflatoxin B1 concentration of 2.5ppb and a CV value of 3.3%.

[0033] Fig.11It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for aflatoxin B1 concentration of 5ppb and the CV value is 3.5%.

[0034] Fig.12 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for aflatoxin B1 concentration of 10ppb and a CV value of 5.2%.

[0035] Fig.13 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / domitoxin, which can detect the stability of aflatoxin B1 at a concentration of 20ppb and a CV value of 4.5%.

[0036] Fig.14 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. The recovery rate of aflatoxin B1 at a concentration of 5ppb is 85.9%.

[0037] Fig.15 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. The recovery rate of aflatoxin B1 at a concentration of 10ppb is 86.6%.

[0038] Fig.16 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. The recovery rate of aflatoxin B1 at a concentration of 15ppb is 94.2%.

[0039] Fig.17 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. The recovery rate of aflatoxin B1 at a concentration of 20ppb is 85.2%.

[0040] Fig.18 It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / domitoxin, and shows the color development of zearalenone ZEN at different added concentrations.

[0041] Fig.19 Read using a fluorescent immunoassay analyzer Fig.18 Detect the T and C line values ​​of the card, calculate the T / C ratio, and draw a curve of T / C and zearalenone ZEN concentration.

[0042] Fig. 20 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for zearalenone ZEN concentration of 0 and the CV value is 3.1%.

[0043] Fig.21 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for zearalenone ZEN concentration of 10ppb and the CV value is 4.2%.

[0044] Fig. 22 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for zearalenone ZEN concentration of 25ppb and a CV value of 2.8%.

[0045] Fig.23 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for zearalenone ZEN concentration of 50ppb and the CV value is 4.5%.

[0046] Fig.24 It is a three-in-one fluorescence quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for zearalenone ZEN concentration of 100ppb and the CV value is 1.7%.

[0047] Fig.25 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for zearalenone ZEN concentration of 200ppb and the CV value is 2.2%.

[0048] Fig.26 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for zearalenone ZEN concentration of 500ppb and a CV value of 3.4%.

[0049] Fig. 27 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. The recovery rate of zearalenone ZEN at a concentration of 10ppb is 85.6%.

[0050] Fig.28 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. The recovery rate of zearalenone ZEN at a concentration of 50ppb is 90.4%.

[0051] Fig.29 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. The recovery rate of zearalenone ZEN at a concentration of 100ppb is 85.1%.

[0052] Fig.30It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. The recovery rate of zearalenone ZEN at a concentration of 200ppb is 86.5%.

[0053] Fig.31 It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / deoxynivalenol, and shows the color development of different added concentrations of deoxynivalenol DON.

[0054] Fig.32 Read using a fluorescent immunoassay analyzer Fig.33 Detect the T and C line values ​​of the card, calculate the T / C ratio, and draw a curve between T / C and DON concentration.

[0055] Fig.33 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for the concentration of vomitoxin DON of 0, with a CV value of 3.7%.

[0056] Fig.34 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for vomitoxin DON concentration of 10, with a CV value of 3.1%.

[0057] Fig.35 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for vomitoxin DON concentration of 50, with a CV value of 0.8%.

[0058] Fig.36 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for vomitoxin DON concentration of 100, with a CV value of 3%.

[0059] Fig.37 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for the concentration of vomitoxin DON at 200, with a CV value of 2.4%.

[0060] Fig.38 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for vomitoxin DON concentration of 500 and the CV value is 3.2%.

[0061] Fig.39 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. It is a stability test for vomitoxin DON concentration of 1000 with a CV value of 3.5%.

[0062] Fig.40It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. The recovery rate of the test for the concentration of vomitoxin DON is 10ppb, and the recovery rate is 85.1%.

[0063] Fig.41 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. The recovery rate of the test for DON concentration of 50ppb is 85.6%.

[0064] Fig.42 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. The recovery rate of vomitoxin DON concentration is 100ppb, and the recovery rate is 86.1%.

[0065] Fig.43 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. The recovery rate of vomitoxin DON at a concentration of 200ppb is 85.8%.

[0066] Fig.44 It is a three-in-one fluorescent quantitative test card for zearalenone / aflatoxin B1 / vomitoxin. The recovery rate of vomitoxin DON concentration is 500ppb, and the recovery rate is 88.6%. DETAILED DESCRIPTION

[0067] In order to further explain the technical means and effects of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods, but the present invention is not limited to the scope of the embodiments. The reagents and instruments involved in the embodiments of the present invention are as follows:

[0068] (1) Reagents and consumables

[0069]

[0070]

[0071] (2) Instruments and Equipment

[0072]

[0073]

[0074] Experimental methods without specific conditions specified in the examples are usually performed according to the conditions recommended by the instrument or reagent manufacturers.

[0075] Example 1 Preparation of fluorescence detection card

[0076] 1. Reagent Preparation

[0077] 150mmol / l MES activation buffer: Dissolve 9.76g in deionized water and make up to 1L;

[0078] 250mg / ml EDC: Weigh 50mg EDC, add 1ml MES and dissolve thoroughly.

[0079] 350mg / ml NHS: weigh 50mg NHS, add 1ml MES and dissolve thoroughly;

[0080] 440mmol / l boric acid borax coupling buffer: Dissolve 1.15g borax and 1.73g boric acid in deionized water and adjust the volume to 1L;

[0081] Blocking solution: 10% glycine + 10% BSA

[0082] Microsphere storage solution: 10mmol / l Tris, pH 8.5, 1% BSA, 2% sucrose, 2% trehalose

[0083]

[0084] Sample pad treatment solution: Take 10g BSA and 5mL Tween-20 respectively and dissolve them in 1L 10mM PBS

[0085]

[0086] 2. Operation steps

[0087] 2.1 Microsphere pretreatment

[0088] 2.1.1Use an ultrasonic cleaner to ultrasonically clean the fluorescent microspheres in an ice-water bath for 3 minutes. The ratio of microspheres to MES buffer is 1:100. Centrifuge at 13,300 rpm for 15 minutes, remove the supernatant, add 1 mL MES buffer, and resuspend in an ice-water bath using an ultrasonic cleaner. Repeat this process 3 times. Finally, use 0.5 mL MES buffer to ultrasonically resuspend, and use an ultrasonic disruptor to ultrasonically clean in an ice-water bath for 3 minutes.

[0089] 2.2 Activation

[0090] 2.2.11. Take 10uL of microspheres and add them to 1mL of activation buffer. Centrifuge at 13300g for 25 minutes and discard the supernatant.

[0091] 2. Activation: resuspend with 500uL activation buffer by ultrasonication, add 25μL each of 50mg / mL LEDC and NHC, activate for 15 minutes, centrifuge at 13300 for 25 minutes, and discard the supernatant.

[0092] 3. Coupling: resuspend with 500uL coupling buffer by ultrasonication, centrifuge and discard the supernatant, resuspend with 500uL coupling buffer by ultrasonication, add 30ug antibody diluted with coupling buffer, and mix on a horizontal shaker for 2h.

[0093] 4. Blocking: After the reaction is completed, centrifuge and discard the supernatant, add 1 mL of blocking solution, resuspend by ultrasound, and block overnight.

[0094] 5. Storage: After sealing, centrifuge and discard the supernatant, re-dissolve and resuspend to 100uL by ultrasonication.

[0095] 2.6 Continue the experiment

[0096] Film cutting

[0097] The 4 lines are

[0098] C:1mg / mL

[0099] Zen:1:2

[0100] B1: 1:2

[0101] Don:1:2

[0102] The spray ball is 6ul / cm

[0103] Example 2 Sensitivity Test

[0104] The sensitivity test of the fluorescence detection card prepared in Example 1 was carried out. The results are as follows: Figure 1-3 shown.

[0105] Figure 1 It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / domitoxin. The line can be eliminated when the concentration of zearalenone ZEN is 150ppb.

[0106] Figure 2 It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / domitoxin, which can eliminate the line when the concentration of aflatoxin B1 is 20ppb.

[0107] Figure 3 It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / deoxynivalenol. It can eliminate the line when the concentration of deoxynivalenol DON is 500ppb.

[0108] Example 3 Stability Test

[0109] The stability test of the fluorescence detection card prepared in Example 1 was carried out. The results are as follows: Figure 6-13 , Figure 20-26 , Figure 33-39 shown.

[0110] Figure 6, Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11 , Fig.12 , Fig.13 It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / deoxynivalenol. The concentrations of aflatoxin B1 are 0ppb, 0.05ppb, 0.5ppb, 1ppb, 2.5ppb, 5ppb, 10ppb, and 20ppb, respectively. The CV values ​​are all lower than 5%, with good stability.

[0111] Fig. 20 , Fig.21 , Fig. 22 , Fig.23 , Fig.24 , Fig.25 , Fig.26 It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / deoxynivalenol. The concentrations of aflatoxin B1 are 0ppb, 10ppb, 25ppb, 50ppb, 100ppb, 200ppb, and 500ppb, respectively. The CV values ​​are all lower than 5%, with good stability.

[0112] Fig.33 , Fig.34 , Fig.35 , Fig.36 , Fig.37 , Fig.38 , Fig.39 It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / vomitoxin. The concentrations of aflatoxin B1 are 0ppb, 10ppb, 50ppb, 100ppb, 200ppb, 500ppb, and 1000ppb, respectively. The CV values ​​are all lower than 5%, with good stability.

[0113] Example 4 Recovery Test

[0114] The recovery rate of the fluorescence detection card prepared in Example 1 was tested. The results are as follows: Figure 14-17 , Figure 27-30 , Figure 40-44 shown.

[0115] Fig.14 , Fig.15 , Fig.16 , Fig.17 It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / vomitoxin. The concentrations of aflatoxin B1 are 5ppb, 10ppb, 15ppb, and 20ppb, respectively, and the recovery rates are all higher than 85%.

[0116] Fig. 27 , Fig.28, Fig.29 , Fig.30 It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / vomitoxin. The concentrations of zearalenone ZEN are 10ppb, 50ppb, 100ppb, and 200ppb, respectively, and the recovery rates are all higher than 85%.

[0117] Fig.40 , Fig.41 , Fig.42 , Fig.43 , Fig.44 It is a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / vomitoxin. The concentrations of vomitoxin DON are 10ppb, 50ppb, 100ppb, 200ppb, and 500ppb, respectively, and the recovery rates are all higher than 85%.

[0118] In summary, the present invention provides a three-in-one fluorescent quantitative detection card for zearalenone / aflatoxin B1 / deoxynivalenol. The detection sensitivity of zearalenone can reach 150 ppb, the sensitivity of aflatoxin B1 can reach 20 ppb, and the sensitivity of deoxynivalenol can reach 500 ppb. The national detection standards are 60 ppb for zearalenone, 20 ppb for aflatoxin B1, and 500 ppb for deoxynivalenol, respectively. The sensitivity of the fluorescent quantitative detection card is higher than the national standard.

[0119] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A fluorescent immunoassay card, comprising a sample pad, a labeling pad, a nitrocellulose membrane and an absorption pad arranged in sequence, characterized in that: The marking pad is sprayed with a monoclonal antibody solution marked with fluorescent microspheres, and the monoclonal antibody solution is a zearalenone-specific monoclonal antibody, aflatoxin-specific monoclonal antibody, and vomitoxin-specific monoclonal antibody.

2. The fluorescent immunoassay card according to claim 1, characterized in that: The concentration of ZEN (zearalenone-specific monoclonal antibody) is 4-7 mg / mL, preferably 5-6 mg / mL; Preferably, the concentration of AFB1 (aflatoxin-specific monoclonal antibody) is 3-6 mg / mL, preferably 4-5 mg / mL; Preferably, the concentration of DON (deoxyribonucleic acid-specific monoclonal antibody) is 3-6 mg / mL, preferably 4-5 mg / mL.

3. The fluorescent immunoassay card according to claim 2, characterized in that: A detection line and a quality control line are arranged in parallel on the nitrocellulose membrane, and the detection line is coated with a corresponding protein that reacts with the sample to be tested by antigen-antibody reaction; Preferably, the test line is coated with zearalenone-BSA, aflatoxin B1-BSA, and vomitoxin-BSA; Preferably, the concentration of zearalenone-BSA is 2-5 mg / mL, preferably 3-4 mg / mL; Preferably, the concentration of aflatoxin B1-BSA is 2-5 mg / mL, preferably 3-4 mg / mL; Preferably, the concentration of DON-BSA is 1-4 mg / mL, preferably 2-3 mg / mL.

4. The fluorescent immunoassay card according to any one of claims 1 to 3, characterized in that: The quality control line is coated with a corresponding antigen or antibody that undergoes an antigen-antibody binding reaction with the labeled antibody or antigen; Preferably, the quality control line is coated with anti-mouse IgG antibody, anti-rabbit IgG antibody or anti-chicken IgG antibody, preferably anti-mouse IgG antibody, and the quality control line is coated with goat anti-mouse secondary antibody.

5. The fluorescent immunoassay card according to claim 1, characterized in that: The width of the sample pad and the absorption pad is about 20-30 mm.

6. The fluorescent immunoassay card according to claim 1, characterized in that: The width of the marking pad is about 8-12 mm.

7. The fluorescent immunoassay card according to claim 1, characterized in that: The width of the fluorescent immunoassay card is 2-5 mm.

8. A detection kit, characterized in that: It comprises a fluorescent immunoassay card as described in any one of claims 1 to 7.

9. A method for detecting a test strip, characterized in that: Using the test strip according to any one of claims 1 to 7, using the kit according to claim 8, the detection method comprises the following steps: Add the sample to be tested to the spotting well, react with immunochromatography for 5-10 minutes, and observe the color change.

10. Use of the fluorescent immunoassay card according to any one of claims 1 to 5 in detecting multiple mycotoxins in cereals.

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