Toxin-producing ability index molecule of aflatoxin-producing fungus and its use

Abstract

The present invention relates to an indicator molecule for the toxin production ability of aflatoxin-producing bacteria and uses thereof. The amino acid sequence of the toxin production ability indicator molecule AFT-YJFZ01 for aflatoxin-producing bacteria is shown in SEQ ID NO: 1. The indicator molecule of the present invention is used to identify the aflatoxin production ability of aflatoxin-producing strains of the genus Aspergillus and to identify the presence or absence of aflatoxin-producing bacteria with strong toxin production ability in agricultural land, agricultural products, and feed, and is easy to disseminate and use.

Description

Technical Field

[0001] The present invention relates to a toxin-producing ability index molecule of an aflatoxin-producing bacterium and its use.

Background Art

[0002] Aflatoxin is a highly toxic and harmful contaminant that is the most common type of contaminant in food. In recent years, the overall pollution trend has been increasing, seriously threatening food safety and people's health. Aflatoxin is the most toxic mycotoxin in nature, and aflatoxin B1 is a Class I carcinogen recognized by the International Agency for Research on Cancer (IARC). It has caused many poisoning incidents in humans and animals and is one of the main reasons for the high incidence of liver cancer. According to the statistics of Web of Science search data in the past five years, the types of foods and raw materials contaminated with aflatoxin exceed 110, occupying the top of the contaminants. However, there are still no research examples at home and abroad that can give early warnings at the molecular level for contamination by microbial toxins such as aflatoxin in advance, and it is difficult to meet the urgent need for early warnings.

[0003] Conventional early warning methods for aflatoxins are mainly established based on aflatoxin detection technology and are used for the evaluation of toxin contamination levels or post-harvest contamination levels and the assessment of intake risks. However, once detected, contamination has often already occurred, making it difficult to meet the urgent requirements for prior early warning, prevention, and control guidance. The European Rapid Alert System for Food and Feed (RASFF) uses tolerance levels and detection to obtain the aflatoxin content in food and feed and issues early warnings for food and feed imported by various countries into the EU. U.S. research institutions have established early warning models such as multivariate logistic regression analysis and superimposed Gaussian processing based on aflatoxin detection technology and contamination monitoring data, which are mainly used to evaluate the mycotoxin contamination levels and intake risks of agricultural products such as post-harvest corn.

[0004] Summarizing the research progress at home and abroad in the past 20 years, the current lack of early warning molecules for aflatoxins is the fundamental reason. To address this bottleneck problem, the inventors have conducted research for more than 10 years to construct the Chinese aflatoxin-producing strain library, the strain toxin-producing capacity database, and the strong toxin-producing strain protein antibody library, and have established an antibody library method for discovering the toxin-producing capacity index molecules of aflatoxin strains, successfully inventing the toxin-producing capacity index molecules of aflatoxin-producing bacteria, which are used to identify the aflatoxin-producing ability of strains, discover whether there are aflatoxin-producing strains with strong toxin-producing ability in farmland, agricultural products, and feed, and provide a scientific basis for the timely detection and early control of aflatoxin contamination risks.

Summary of the Invention

Problems to be Solved by the Invention

[0005] In view of the deficiencies existing in the prior art, the present invention provides a toxin-producing ability index molecule AFT-YJFZ01 of aflatoxin-producing bacteria and a method for identifying the toxin-producing ability of aflatoxin-producing bacteria in the system. It is used to identify the aflatoxin-producing ability of Aspergillus strains and to determine whether there are aflatoxin-producing strains with strong toxin-producing ability in farmland, agricultural products and feeds, and is easy to popularize and use.

Means for Solving the Problems

[0006] In order to solve the above-mentioned technical problems, the present invention adopts the following technical means.

[0007] The amino acid sequence of the toxin-producing ability index molecule AFT-YJFZ01 of aflatoxin-producing bacteria provides the toxin-producing ability index molecule AFT-YJFZ01 of aflatoxin-producing bacteria shown in SEQ ID NO: 1.

[0008] Based on the detected content of the toxin-producing ability index molecule AFT-YJFZ01 of aflatoxin-producing bacteria, the toxin-producing ability of aflatoxin-producing bacteria in the system is identified, and the amino acid sequence of the toxin-producing ability index molecule AFT-YJFZ01 of aflatoxin-producing bacteria is shown in SEQ ID NO: 1, and a method for identifying the toxin-producing ability of aflatoxin-producing bacteria in the system is provided.

[0009] Specifically, by using the amino acid sequence or a partial sequence thereof of the toxin-producing ability index molecule AFT-YJFZ01 of aflatoxin-producing bacteria, an antibody corresponding to the protein is prepared according to the conventional antibody preparation procedure to realize the quantitative detection of the index molecule protein, and by other detection technical means, the quantitative detection having a one-to-one correspondence with these proteins is realized, whereby the above-mentioned use can be achieved. The partial sequence means a part of the entire sequence having a one-to-one correspondence with the protein of the index molecule.

[0010] Use for identifying the aflatoxin-producing ability of Aspergillus aflatoxin-producing strains, wherein the higher the content of the toxin-producing force index molecule AFT-YJFZ01 of aflatoxin-producing bacteria in the strain to be identified, the stronger the aflatoxin-producing ability of the strain to be identified, indicating that the toxin-producing force is stronger.

[0011] Specific usage methods are as follows: (1) Providing a nanobody or monoclonal antibody of the toxin-producing force index molecule AFT-YJFZ01 of aflatoxin-producing bacteria; (2) Providing a polyclonal antibody of the toxin-producing force index molecule AFT-YJFZ01 of aflatoxin-producing bacteria; (3) Preparation of the test solution of the strain to be identified: Culturing the strain to be identified, diluting it, and obtaining the test solution of the strain to be identified, Specifically, culturing the strain to be identified in a conventional Czapek medium or other medium suitable for the growth of other strains, with the environmental temperature for culturing being 15-35 °C, the culturing time being 12 h or more, thoroughly homogenizing the mixture of the medium and the culture, and further diluting it 1-10 times with sterile water to obtain the test solution of the strain to be identified; (4) Measuring the aflatoxin-producing ability of the strain to be identified. Specifically, adopting the indirect non-competitive double antibody sandwich method to identify the aflatoxin-producing ability of Aspergillus aflatoxin-producing strains, Coating the nanobody or monoclonal antibody of AFT-YJFZ01 on the immunoplate and washing the plate; Adding the blocking solution for blocking and washing the plate; Adding the test solution for reaction and washing the plate; Adding the polyclonal antibody of AFT-YJFZ01 for reaction and washing the plate; Adding a horseradish peroxidase-labeled antibody that binds to the specific polyclonal antibody of the toxin-producing force index molecule AFT-YJFZ01 of aflatoxin-producing bacteria for reaction and washing the plate; The step of adding a coloring solution and reacting; The step of adding a stop solution, reading with a microplate reader, calculating, obtaining the content of the toxin-producing force index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium, and indicating that the higher the content of the toxin-producing force index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium in the test solution of the strain to be discriminated, the stronger the aflatoxin-producing ability of the aflatoxin toxin-producing strain. It includes.

[0012] Specifically, step (4) is step a: Mix a coating solution of 0.2 to 8.0 μg / mL with the nanobody or monoclonal antibody of the above AFT-YJFZ01 and an ELISA coating buffer, add it to an immunoplate (200 μL / well), and leave it at 4°C overnight or at 37°C for 2 h or more. Discard the coating solution in the above immunoplate, wash the immunoplate with a conventional washing solution for ELISA, and then use skim milk powder with a concentration of 1% or more as a blocking solution (add 300 μL to each well), leave it at room temperature or block it at 37°C for 1 h or more, then discard the blocking solution, and wash the immunoplate with a conventional washing solution for ELISA. Step b: Next, appropriately dilute the test solution of the strain to be discriminated with a conventional phosphate buffer with a pH close to neutral, add it to the wells of the immunoplate (add 200 μL to each well), leave it at room temperature or block it at 37°C for 1 h or more, then discard the liquid, and wash the immunoplate with a conventional washing solution for ELISA. Step c: Next, appropriately dilute the polyclonal antibody of the above AFT-YJFZ01 with a conventional phosphate buffer with a pH close to neutral, add it to the wells of the immunoplate (add 200 μL to each well), leave it at room temperature or block it at 37°C for 1 h or more, then discard the liquid, and wash the immunoplate with a conventional washing solution for ELISA. Step d: Next, dilute the commercially available horseradish peroxidase-labeled antibody with a conventional phosphate buffer with a pH close to neutral as needed, add it to the wells of the immunoplate (add 200 μL to each well), leave it at room temperature or block it at 37 °C for 1 h or more, then discard the liquid, wash the immunoplate with a conventional washing solution for ELISA, Step e: Next, add a conventional chromogenic solution and stop solution for ELISA in sequence, and finally read and calculate the result of the AFT-YJFZ01 content by a microplate reader.

[0013] According to the above technical means, a calibration curve is created using a solution of the toxin-producing ability index molecule AFT-YJFZ01 with a stepwise concentration gradient, and the content of AFT-YJFZ01 is obtained using the calibration curve based on the result of the microplate reader. That is, in step b, the test solution of the strain to be discriminated is replaced with a solution of the toxin-producing ability index molecule AFT-YJFZ01 with a stepwise concentration gradient to create a calibration curve.

[0014] It is used to discriminate whether there is an aflatoxin-producing strain with strong toxin-producing ability in a sample, and the steps of the specific usage method are as follows. (1) Provide a nanobody or monoclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin-producing bacterium. (2) Provide a polyclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin-producing bacterium. (3) Preparation of the sample to be discriminated: Cultivate, dilute the sample to be discriminated to obtain a test solution of the sample to be discriminated. Specifically, weigh the sample to be discriminated, transfer it to sterile water, shake it uniformly at room temperature to produce a uniformly dispersed solution of the test sample, add 10 - 1000 μL of the uniformly dispersed test sample solution to 6 - 600 mL of a conventional Czapek medium or other medium suitable for the growth of aflatoxin-producing bacteria, shake and culture at 15 - 35 °C at 200 ± 50 rpm, sample after culturing for 6 - 24 h to form a test solution of the sample to be discriminated. Determining whether or not there is an aflatoxin toxin-producing strain with strong toxin-producing ability in the sample. Specifically, coating the immuno plate with the nanobody or monoclonal antibody of AFT-YJFZ01 and washing the plate; Adding a blocking solution for blocking and washing the plate; Adding the test sample solution for reaction and washing the plate; Adding the polyclonal antibody of AFT-YJFZ01 for reaction and washing the plate; Adding a horseradish peroxidase-labeled antibody that binds to the polyclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium for reaction and washing the plate; Adding a chromogenic solution for reaction; Adding a stop solution, reading with a microplate reader, calculating, and obtaining the content of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium. Specifically, step (4) includes step a: formulating a coating solution of 0.2 - 8.0 μg / mL with the above-mentioned nanobody or monoclonal antibody of AFT-YJFZ01 and an ELISA coating buffer, adding it to an immuno plate (200 μL / well), leaving it at 4°C overnight or leaving it at 37°C for 2 h or more, discarding the coating solution in the immuno plate, washing the immuno plate with a conventional washing solution for ELISA, then using skim milk powder with a concentration of 1% or more as a blocking solution (adding 300 μL to each well), leaving it at room temperature or blocking it at 37°C for 1 h or more, then discarding the blocking solution and washing the immuno plate with a conventional washing solution for ELISA. Step b: Next, appropriately diluting the test solution of the sample with a conventional phosphate buffer with a pH close to neutral, adding it to the wells of the immuno plate (adding 200 μL to each well), leaving it at room temperature or blocking it at 37°C for 1 h or more, then discarding the liquid and washing the immuno plate with a conventional washing solution for ELISA. Step c: Next, appropriately dilute the polyclonal antibody of AFT-YJFZ01 with a conventional phosphate buffer with a pH close to neutral, add it to the wells of the immunoplate (add 200 μL to each well), leave it at room temperature or block it at 37 °C for 1 h or more, then discard the liquid and wash the immunoplate with a conventional washing solution for ELISA. Step d: Next, appropriately dilute a commercially available horseradish peroxidase-labeled antibody with a conventional phosphate buffer with a pH close to neutral as needed, add it to the wells of the immunoplate (add 200 μL to each well), leave it at room temperature or block it at 37 °C for 1 h or more, then discard the liquid and wash the immunoplate with a conventional washing solution for ELISA. Step e: Next, sequentially add a conventional chromogenic solution and a stop solution for ELISA, and finally read and calculate the result of the AFT-YJFZ01 content by a microplate reader. (5) Evaluate the identification result. The higher the content of the toxin-producing force index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacteria in the sample to be identified, the stronger the toxin-producing force, which is the aflatoxin-producing ability of the strain to be identified. Based on the calculation result of the microplate reader, obtain the content of the toxin-producing force index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacteria per unit volume of the sample test solution, and determine whether the sample to be identified contains a strain with strong aflatoxin toxin-producing force.

[0015] According to the above technical means, the sample to be identified is soil, traditional Chinese medicine materials, agricultural products, feed, etc.

[0016] According to the above technical means, a calibration curve is created using a solution of the toxin-producing force indicator molecule AFT-YJFZ01 with a stepwise concentration gradient, and the content of AFT-YJFZ01 is obtained using the calibration curve based on the results of a microplate reader. That is, in step b, the sample solution to be discriminated is replaced with a solution of the toxin-producing force indicator molecule AFT-YJFZ01 with a stepwise concentration gradient to create a calibration curve. According to the above technical means, the polyclonal antibody of the toxin-producing force indicator molecule AFT-YJFZ01 of the aflatoxin-producing bacterium is different from the animal source of the nanobody or monoclonal antibody of the toxin-producing force indicator molecule AFT-YJFZ01 of the aflatoxin-producing bacterium. Specifically, the nanobody or monoclonal antibody of the toxin-producing force indicator molecule AFT-YJFZ01 of the aflatoxin-producing bacterium can be obtained by the following method. Using AFT-YJFZ01 as an immunizing antigen, alpacas or Balb / c mice are immunized by a conventional method, and further, it can be obtained by using the technical means for preparing known conventional nanobodies or mouse-derived monoclonal antibodies.

[0017] The polyclonal antibody of the toxin-producing force indicator molecule AFT-YJFZ01 of the aflatoxin-producing bacterium can be obtained by the following method. Using AFT-YJFZ01 as an immunizing antigen, experimental rabbits such as New Zealand white rabbits are immunized by a conventional method, and further, a rabbit-derived polyclonal antibody of the toxin-producing force indicator molecule AFT-YJFZ01 of the aflatoxin-producing bacterium can be obtained by using the technical means for preparing known conventional polyclonal antibodies.

[0018] The horseradish peroxidase-labeled antibody that binds to the polyclonal antibody of the toxin-producing force indicator molecule AFT-YJFZ01 of the aflatoxin-producing bacterium is a horseradish peroxidase-labeled goat anti-rabbit antibody, which can be obtained directly by purchasing the product.

[0019] The toxin-producing ability of Aspergillus flavus strains is an indicator for measuring the aflatoxin-producing ability of the strains. The stronger the toxin-producing ability of the strains, the more aflatoxin the strains can produce under the same time and culture conditions. The present invention provides a toxin-producing ability indicator molecule AFT-YJFZ01 for aflatoxin-producing bacteria, and utilizes the characteristic that the content of the toxin-producing ability indicator molecule AFT-YJFZ01 of aflatoxin-producing bacteria has a positive correlation with the toxin-producing ability of the strains. Furthermore, it discriminates the aflatoxin-producing ability of Aspergillus aflatoxin-producing strains, and provides a use for discriminating whether there are aflatoxin-producing strains with strong toxin-producing ability in farmland, agricultural products and feeds. It is practical and easy to popularize, provides an important starting point and scientific basis for the timely detection and early control of aflatoxin contamination risks, promotes the high-quality development of the agricultural industry, and has important significance in ensuring food safety.

Advantages of the Invention

[0020] The beneficial effects of the present invention are as follows. 1. It is used to discriminate the aflatoxin-producing ability of Aspergillus aflatoxin-producing strains. 2. It is used to discriminate whether there are aflatoxin-producing strains with strong toxin-producing ability in farmland, agricultural products and feeds. 3. The operation is simple, the practicability is high, and it is easy to popularize and use. 4. It promotes the high-quality development of the agricultural industry and has important significance in ensuring food safety.

Modes for Carrying Out the Invention

[0021] (Example 1) Preparation of the toxin-producing ability indicator molecule AFT-YJFZ01 of aflatoxin-producing bacteria By formulating 3% (w / v) sucrose, 0.3% (w / v) NaNO3, 0.1% (w / v) K2HPO4, 0.05% (w / v) MgSO4·7H2O, 0.05% (w / v) KCl, 0.001% (w / v) FeSO4 with a pH of 6.5, a Czapek medium was obtained. Ten strains such as the toxin-producing strains HLJ-1, HeNZY-2, HuBha-24, JXZS-29-2, LNct-6, GXfc-34, GDZJ-122-2, JSnt-1, HuNdx-7, HBHA-8-17, etc. published in the public literature "Research on the Distribution, Toxin Productivity and Infection of Aflatoxin in Typical Peanut Production Areas in China" (Master's thesis of the Chinese Academy of Agricultural Sciences, author Zhang Xing, page 33) were randomly selected, each was inoculated into the above Czapek medium, cultured at 28°C at 200 rpm / min for 5 days, then thoroughly homogenized by the conventional method, the cells were disrupted, and the toxin productivity index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacteria was purified by methods such as the conventional protein purification system, protein electrophoresis, and immunoaffinity. As can be seen from the test results, AFT-YJFZ01 can be prepared from the above toxin-producing strain cultures. Under the same culture conditions, the amount of AFT-YJFZ01 prepared from HBHA-8-17 is the largest, and the amount of AFT-YJFZ01 prepared from HLJ-1 is the smallest.

[0022] Discovery method for initial acquisition of the toxin productivity index molecule AFT-YJFZ01 of aflatoxin toxin-producing bacteria: The method for discovering the toxin productivity index molecule AFT-YJFZ01 of aflatoxin strains is as follows: (1) Collect aflatoxin strains with strong toxin productivity, culture them to obtain strain cultures and extracellular secreted protein mixtures, then disrupt the cells of the strain cultures to obtain intracellular protein mixtures, mix the above extracellular secreted protein mixtures and intracellular protein mixtures, add carbodiimide and couple them to obtain aflatoxin antigens; (2) Immunize the above aflatoxin antigens to experimental animals to obtain a nanobody library or a monoclonal antibody library; (3) Obtain a mixed protein solution of aflatoxin strains with different toxin-producing abilities, and use the antibodies in the antibody library obtained in step (2) to detect the proteins of aflatoxin strains with different toxin-producing abilities, and obtain a series of detection signals. (4) Discover an antibody of an aflatoxin strain toxin-producing ability index molecule, which is a nanobody or monoclonal antibody whose detection signal shows a positive correlation with the toxin-producing ability of the above aflatoxin strain, and the protein corresponding to the antibody of the aflatoxin strain toxin-producing ability index molecule is the discovered aflatoxin strain toxin-producing ability index molecule.

[0023] In the above technical means, the aflatoxin strain with strong toxin-producing ability in step (1) is isolated and identified from nature by a conventional method or obtained by artificial transformation, and its toxin-producing ability is 10 μg / kg or more as identified by the NY / T 2311-2013 standard method.

[0024] There are 3 or more aflatoxin strains with different toxin-producing abilities described in step (3), and their toxin-producing abilities showed at least three levels of high, medium, and low as identified by the NY / T 2311-2013 standard method.

[0025] The medium used for culturing the aflatoxin strain with strong toxin-producing ability is Czapek medium or other nutrients for normal growth of aflatoxin, the culture time is 12 h or more, and the environmental temperature for culture is 15-35 °C.

[0026] The cell disruption of the strain culture is performed by methods such as conventional liquid nitrogen grinding or cell disruptors.

[0027] The usage amount of the carbodiimide is 0.005-0.1 g of carbodiimide added per 1.0 milliliter in total of the extracellular secreted protein mixture and the intracellular protein mixture.

[0028] The coupling reaction means reacting at 15-37°C for 2-6 h and then reacting overnight at 4-10°C.

[0029] The immunization is a conventional immunization method and inoculates the aflatoxin antigen. The experimental animals mean mice or alpacas or other experimental animals having similar effects.

[0030] According to the above technical means, the antibody preparation procedure refers to a conventional nanobody preparation technical procedure or a hybridoma monoclonal antibody preparation technical procedure based on conventional cell fusion.

[0031] According to the above technical means, detecting the proteins of aflatoxin strains with different toxin-producing abilities means using the conventional Western Blot technical procedure, that is, transferring the proteins of aflatoxin strains with different toxin-producing abilities to a nitrocellulose membrane, and using the antibodies in the above antibody library to detect by a direct method or an indirect method or other technical procedures having similar effects.

[0032] According to the above technical means, the direct method means binding the antibodies in the above antibody library to a signal substance by a conventional method and performing an immunological binding reaction with the corresponding proteins transferred to the above nitrocellulose membrane.

[0033] According to the above technical means, the indirect method means performing an immunological binding reaction between the antibodies in the above antibody library and the corresponding proteins transferred to the nitrocellulose membrane, and then performing an immunological binding reaction between the antibodies bound to the above nitrocellulose membrane and the second antibody and a signal transduction complex.

[0034] The signal substance in the above detection is horseradish peroxidase or gold colloid or fluorescent substance or other substances having similar effects. The detection signal is a color reaction signal or a dot signal or a fluorescent signal.

[0035] (Example 2) Preparation of Nanobody of Toxin-producing Force Index Molecule AFT-YJFZ01 of Aflatoxin-producing Bacteria Using AFT-YJFZ01 as an immunogen, alpacas or Balb / c mice can be immunized by conventional methods, and can be obtained by further using technical means for preparing known conventional nanobodies or mouse-derived monoclonal antibodies.

[0036] Dissolve the AFT-YJFZ01 prepared above in a conventional PBS buffer or physiological saline until the concentration reaches 0.1 mg / mL or more, mix and emulsify it with an equal volume of Freund's complete adjuvant, and immunize alpacas by injecting multiple points subcutaneously or intradermally in the back. After that, boost immunization is performed once every 2 to 4 weeks, and Freund's complete adjuvant is replaced with Freund's incomplete adjuvant during boost immunization. The immune effect is monitored using a conventional ELISA process until the serum titer of alpacas no longer increases. Then, venous blood of immunized alpacas is collected, and operations such as extraction of total RNA, synthesis of cDNA, amplification of VHH gene, recovery of VHH gene fragment, ligation of VHH gene and double-digested pCANTAB 5 E(his) vector, electrotransformation of ligation product, construction of nanobody gene library and rescue of nanobody gene library are completed with reference to the method of Patent Document CN103866401A, and finally, the rescued nanobody gene library is obtained.

[0037] The prepared AFT-YJFZ01 above was immobilized on a solid-phase carrier such as a 96-well immunoplate at gradients of 8 μg / well, 2 μg / well, 0.5 μg / well, and 0.1 μg / well. Referring to the method of Patent Document CN103866401A, the rescued nanobody gene library above was panned 2 to 4 times. Furthermore, the antibodies produced by each phage clone were identified using AFT-YJFZ01 and indirect non-competitive ELISA. The phage corresponding to the positive result was the phage positive clone. Furthermore, this positive clone was used to prepare an AFT-YJFZ01 nanobody, which is a nanobody, by the conventional method of nanobody preparation. It was further used in the research, preferably a nanobody characterized by the ELISA method, with strong specificity and high affinity.

[0038] (Example 3) Preparation of Monoclonal Antibody Against Toxin Producing Force Index Molecule AFT-YJFZ01 of Aflatoxin Producing Bacteria Using AFT-YJFZ01 as an immunogen, alpacas or Balb / c mice can be immunized by the conventional method, and can be obtained by further using the technical means for preparing known conventional nanobodies or mouse-derived monoclonal antibodies.

[0039] The prepared AFT-YJFZ01 above was dissolved in the conventional PBS buffer or physiological saline until the concentration reached 0.1 mg / mL or higher, mixed and emulsified with an equal volume of Freund's complete adjuvant, and BALB / c mice were immunized by injecting multiple points subcutaneously or intradermally into the back. Subsequently, booster immunizations were performed once every 2 - 4 weeks, and Freund's complete adjuvant was replaced with Freund's incomplete adjuvant during the booster immunization. The immune effect was monitored using the conventional ELISA process until the serum titer of BALB / c mice no longer increased. Subsequently, spleen cells of the immunized mice were isolated, and the spleen cells were fused with mouse-derived myeloma cells SP2 / 0. The selective culture operation of hybridoma cells using a semi-solid medium was completed with reference to the method of Patent Document CN103849604A. After white spots grew at the needle tip in the semi-solid medium, the white spots were taken out into 96-well culture plates containing the conventional medium of hybridoma respectively, thereby obtaining a monoclonal hybridoma resource bank.

[0040] A monoclonal antibody, which is the culture supernatant of the above monoclonal hybridoma, was obtained with reference to the method of Patent Document CN103849604A. AFT-YJFZ01 prepared above was immobilized on a solid phase carrier such as a 96-well immunoplate at gradients of 8 μg / well, 2 μg / well, 0.5 μg / well, and 0.1 μg / well, and each monoclonal antibody was identified using an indirect non-competitive ELISA process. Positive clones were selected to obtain an AFT-YJFZ01 monoclonal antibody, which was further used in the research. Preferably, the AFT-YJFZ01 monoclonal antibody has the characteristics of strong specificity and high affinity by detection.

[0041] (Example 4) Preparation of Rabbit-derived Polyclonal Antibody of Toxin-producing Force Index Molecule AFT-YJFZ01 of Aflatoxin-producing Bacteria Using AFT-YJFZ01 as an immunizing antigen, experimental rabbits such as New Zealand white rabbits can be immunized by the conventional method, and can be obtained by further utilizing the known technical means for preparing conventional rabbit polyclonal antibodies.

[0042] Using the toxin-producing ability index molecule AFT-YJFZ01 of Aspergillus flavus toxin-producing bacteria as an antigen directly, it was mixed and emulsified in equal volume with a solution at a concentration of 0.1 mg / mL or more and Freund's complete adjuvant, and New Zealand white rabbits were immunized by injecting multiple points subcutaneously or intradermally in the back. After that, booster immunizations were performed once every 2 to 4 weeks, and Freund's complete adjuvant was replaced with Freund's incomplete adjuvant during booster immunizations. The immune effect was monitored until the serum titer of the immunized animals no longer increased using the conventional ELISA process, and a rabbit-derived polyclonal antibody against the toxin-producing ability index molecule AFT-YJFZ01 of Aspergillus flavus toxin-producing bacteria, which is the serum of the immunized animals by the conventional method, was prepared.

[0043] (Example 5) Discrimination of the toxin-producing ability of Aspergillus flavus strains using the toxin-producing ability index molecule AFT-YJFZ01 of Aspergillus flavus toxin-producing bacteria First step, preparation of the test solution of the strain to be discriminated: According to the strength of the toxin-producing ability of the strain, 10 strains such as the toxin-producing strains HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, HeNZY-2, HuBha-24, JSnt-1, HuNdx-7, GDZJ-108-19, HBHA-8-17 published in the public literature "Research on the Distribution, Toxin-Producing Ability and Infection of Aspergillus flavus in Typical Peanut Production Areas in China" (Master's thesis of the Chinese Academy of Agricultural Sciences, author Zhang Xing, page 33) were selected. The strain to be discriminated was cultured in the conventional Czapek medium or other media suitable for the growth of Aspergillus flavus toxin-producing bacteria. The environmental temperature for culture was 15 to 35 °C, the culture time was 12 h or more, the mixture of the medium and the culture was homogenized sufficiently, and then diluted 5-fold with sterile water to obtain the test solution of the strain to be discriminated.

[0044] Second step, measurement of the test solution of the strain to be identified: Dissolve the nanobody or monoclonal antibody of AFT-YJFZ01 in a conventional ELISA coating buffer to form a coating solution of 2 μg / mL, and further add it to a 96-well immunoplate at 200 μL / well, and leave it overnight at 4°C or for 2 h or more at 37°C. Discard the coating solution in the immunoplate, and further wash the immunoplate with a conventional washing solution for ELISA. Next, use skim milk powder with a concentration of 1% or more as a blocking solution, add 300 μL to each well, leave it at room temperature or block it at 37°C for 1 h or more, then discard the blocking solution, and wash the immunoplate with a conventional washing solution for ELISA. Next, appropriately dilute the test solution with a conventional phosphate buffer with a pH close to neutral, and further add 200 μL to each well, or add 200 μL of the solution of the toxin-producing ability index molecule AFT-YJFZ01 of the present invention with a stepwise concentration to each well, leave it at room temperature or block it at 37°C for 1 h or more, then discard the liquid, and further wash the immunoplate with a conventional washing solution for ELISA. Next, appropriately dilute the rabbit-derived polyclonal antibody of AFT-YJFZ01 with a conventional phosphate buffer with a pH close to neutral, add 200 μL to each well, leave it at room temperature or block it at 37°C for 1 h or more, then discard the liquid, and wash the immunoplate with a conventional washing solution for ELISA. Next, dilute the commercially available horseradish peroxidase-labeled goat anti-rabbit antibody with a conventional phosphate buffer with a pH close to neutral according to the instructions, add 200 μL to each well, leave it at room temperature or block it at 37°C for 1 h or more, then discard the liquid, and wash the immunoplate with a conventional washing solution for ELISA. Next, add the conventional chromogenic solution and stop solution for ELISA in sequence, and finally read and calculate the AFT-YJFZ01 content with a microplate reader.

[0045] According to the results of the above-mentioned literature "Research on the Distribution, Toxin Productivity and Infection of Aflatoxin in Typical Peanut Production Areas in China", the toxin productivities of 10 strains of bacteria such as HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, HeNZY-2, HuBha-24, JSnt-1, HuNdx-7, GDZJ-108-19, and HBHA-8-17 are in the order of 0 μg / L, 0 μg / L, 3.8 μg / L, 4.9 μg / L, 67.2 μg / L, 81.7 μg / L, 192.0 μg / L, 204.4 μg / L, 297.4 μg / L, and 1027.5 μg / L. HBZHX-21 and HBXY-36 are non-toxin-producing strains, HBHA-1-4 and GDZJ-6 are strains with weak toxin productivity, and HBHA-8-17 and GDZJ-108-19 are strains with strong toxin productivity.

[0046] Step 3, Evaluation of Identification Results: The higher the content of the toxin-producing force index molecule AFT-YJFZ01 of the aflatoxin-producing bacterium in the strain to be identified, the stronger the toxin-producing force of the strain to be identified, which indicates that the toxin-producing force becomes stronger. By the above technical means, the measurement results of the AFT-YJFZ01 content of 10 strains such as HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, HeNZY-2, HuBha-24, JSnt-1, HuNdx-7, GDZJ-108-19, and HBHA-8-17 are that the AFT-YJFZ01 content per milliliter of the medium is 0 ng, 0 ng, 8 ng, 12 ng, 49 ng, 51 ng, 104 ng, 115 ng, 175 ng, and 536 ng. From the results, similarly, HBZHX-21 and HBXY-36 are non-toxin-producing strains, HBHA-1-4 and GDZJ-6 are strains with weak toxin-producing force, and HBHA-8-17 and GDZJ-108-19 are strains with strong toxin-producing force. The measurement results are consistent with the order of the strength of the toxin-producing force of the strains published in the above-mentioned public literature "Research on the Distribution, Toxin-Producing Force and Infection of Aflatoxin in Typical Peanut Production Areas in China", and the identification results of the strains with strong toxin-producing force and the strains with weak toxin-producing force are consistent with the literature, proving that the technical means according to the present invention can identify the toxin-producing force of aflatoxin strains, the method is simple, easy to operate, and highly practical.

[0047] (Example 6) Identification of whether a strain with strong aflatoxin toxin-producing force is contained in agricultural land by using the toxin-producing force index molecule AFT-YJFZ01 of aflatoxin-producing bacteria Step 1, Preparation of the test solution of the sample to be identified: A total of 4 rhizosphere soil samples during the flowering period of peanuts from Jilin, Liaoning, Jiangxi, Fujian, etc. were selected and named as Soil Sample - 1, Soil Sample - 2, Soil Sample - 3, and Soil Sample - 4 in sequence. The soil samples of the test farmland were weighed sequentially, pulverized, transferred to sterile water, with a concentration of 0.5 g / mL, shaken uniformly at room temperature to produce a sample homogeneous dispersion solution in which the test sample was uniformly dispersed. 50 μL of the soil dilution was taken, added to 30 mL of the conventional Sabouraud liquid medium, cultured with shaking at 200 rpm at 28°C, sampled after 24 h of culture, and the test solution of the sample to be identified was formed.

[0048] Step 2, Measurement of the test solution of the sample to be identified: Dissolve the nanobody or monoclonal antibody of AFT-YJFZ01 in a conventional ELISA coating buffer to form a coating solution with a concentration of 0.2 - 8.0 μg / mL. Then, add 200 μL / well to a 96-well immunoplate and leave it overnight at 4°C or for 2 h or more at 37°C. Discard the coating solution in the immunoplate and wash the immunoplate with a conventional washing solution for ELISA. Next, use skim milk powder with a concentration of 1% or more as a blocking solution, add 300 μL to each well, leave it at room temperature or block it at 37°C for 1 h or more. Then, discard the blocking solution and wash the immunoplate with a conventional washing solution for ELISA. Next, appropriately dilute the test solution with a conventional phosphate buffer with a pH close to neutral and add 200 μL to each well, or add 200 μL of the solution of the toxin-producing ability index molecule AFT-YJFZ01 of the present invention with stepwise concentrations (concentrations are 0.00003, 0.0003, 0.003, 0.03, 0.3, 3, 30, 300 ng / mL and are used to obtain a calibration curve) to each well. Leave it at room temperature or block it at 37°C for 1 h or more. Then, discard the liquid and wash the immunoplate with a conventional washing solution for ELISA. Next, appropriately dilute the rabbit-derived polyclonal antibody of AFT-YJFZ01 with a conventional phosphate buffer with a pH close to neutral, add 200 μL to each well, leave it at room temperature or block it at 37°C for 1 h or more. Then, discard the liquid and wash the immunoplate with a conventional washing solution for ELISA. Next, dilute the commercially available horseradish peroxidase-labeled goat anti-rabbit antibody with a conventional phosphate buffer with a pH close to neutral according to the instructions, add 200 μL to each well, leave it at room temperature or block it at 37°C for 1 h or more. Then, discard the liquid and wash the immunoplate with a conventional washing solution for ELISA. Next, add the conventional chromogenic solution and stop solution for ELISA in sequence, finally read it with a microplate reader, and calculate the AFT-YJFZ01 content based on the calibration curve.

[0049] Step 3, Evaluation of the discrimination result: When the content of the toxin-producing force index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium in the sample to be discriminated is high, it indicates that the sample to be discriminated contains strains with strong aflatoxin toxin-producing ability. The results of measuring the AFT-YJFZ01 content in Soil Sample-1, Soil Sample-2, Soil Sample-3, and Soil Sample-4 by the above technical means are that the AFT-YJFZ01 content per milliliter of the culture solution is 0.3 ng, 0.2 ng, 16 ng, and 19 ng in sequence. As can be seen from this result, the AFT-YJFZ01 content per milliliter of the culture solution of Soil Sample-1 and Soil Sample-2 is both below 1.0 ng, does not contain strains with strong aflatoxin toxin-producing ability, and the contamination risk after peanut harvest in the corresponding farmland is very low. The AFT-YJFZ01 content per milliliter of the culture solution of Soil Sample-1 and Soil Sample-2 is both 10 ng or more, contains strains with strong aflatoxin toxin-producing ability, and the contamination risk after peanut harvest in the corresponding farmland is high.

[0050] (Example 7) Discrimination of whether agricultural products using the toxin-producing force index molecule AFT-YJFZ01 of aflatoxin toxin-producing bacteria contain strains with strong aflatoxin toxin-producing ability Step 1, Preparation of the test solution of the sample to be discriminated: A total of 4 agricultural product samples such as peanuts, corns, rice, and wheat were selected, the test farmland soil samples were weighed sequentially, pulverized, transferred to sterile water, and the concentration was set to 0.5 g / mL, and uniformly shaken at room temperature to produce a uniform dispersion of the test sample. 100 μL of the sample uniform dispersion was taken, added to 50 mL of a conventional Sabouraud liquid medium, shake-cultured at 28 °C and 200 rpm, cultured for 6 h, the sample was collected, and the test solution of the sample to be discriminated was formed.

[0051] Second step, measurement of the test solution of the sample to be identified: Dissolve the nanobody or monoclonal antibody of AFT-YJFZ01 in a conventional ELISA coating buffer to form a coating solution with a concentration of 0.2 - 8.0 μg / mL, and further add it to a 96-well immunoplate at 200 μL / well, and leave it overnight at 4°C or for 2 hours or more at 37°C. Discard the coating solution in the immunoplate, and further wash the immunoplate with a conventional washing solution for ELISA. Next, use skim milk powder with a concentration of 1% or more as a blocking solution, add 300 μL to each well, leave it at room temperature or block it at 37°C for 1 hour or more, then discard the blocking solution and wash the immunoplate with a conventional washing solution for ELISA. Next, appropriately dilute the test solution with a conventional phosphate buffer close to neutral pH, and further add 200 μL to each well, or add 200 μL of the solution of the toxin-producing ability index molecule AFT-YJFZ01 of the present invention with stepwise concentrations (the concentrations are 0.00003, 0.0003, 0.003, 0.03, 0.3, 3, 30, 300 ng / mL and are used to obtain a calibration curve) to each well, leave it at room temperature or block it at 37°C for 1 hour or more, then discard the liquid and further wash the immunoplate with a conventional washing solution for ELISA. Next, appropriately dilute the rabbit-derived polyclonal antibody of AFT-YJFZ01 with a conventional phosphate buffer close to neutral pH, add 200 μL to each well, leave it at room temperature or block it at 37°C for 1 hour or more, then discard the liquid and wash the immunoplate with a conventional washing solution for ELISA. Next, dilute the commercially available horseradish peroxidase-labeled goat anti-rabbit antibody with a conventional phosphate buffer close to neutral pH according to the instructions, add 200 μL to each well, leave it at room temperature or block it at 37°C for 1 hour or more, then discard the liquid and wash the immunoplate with a conventional washing solution for ELISA. Next, add the conventional color-developing solution and stopping solution for ELISA in sequence, finally read by a microplate reader, and calculate the AFT-YJFZ01 content based on the calibration curve.

[0052] Step 3, Evaluation of the discrimination result: When the content of the toxin-producing force index molecule AFT-YJFZ01 of the aflatoxin-producing fungus in the sample to be discriminated is high, it indicates that the sample to be discriminated contains strains with strong aflatoxin toxin-producing ability. The results of measuring the content of AFT-YJFZ01 in peanut, corn, rice, and wheat samples by the above technical means are that the content of AFT-YJFZ01 per milliliter of the culture solution is 12 ng, 13 ng, 0.7 ng, and 0 ng in order. As can be seen from this result, the content of AFT-YJFZ01 per milliliter of the culture solution of peanut and corn samples is both 10 ng or more, containing strains with strong aflatoxin toxin-producing ability, with a high contamination risk; the content of AFT-YJFZ01 per milliliter of the culture solution of rice samples is 1.0 or less, not containing strains with strong aflatoxin toxin-producing ability, with a very low contamination risk; the measured content of AFT-YJFZ01 per milliliter of the culture solution of wheat samples is 0, not containing aflatoxin-producing strains, and there is almost no aflatoxin contamination risk.

[0053] (Example 8) Discrimination of whether a feed containing a strong aflatoxin toxin-producing strain contains the toxin-producing force index molecule AFT-YJFZ01 of the aflatoxin-producing fungus Step 1, Preparation of the test solution of the sample to be discriminated: A total of 4 test feed samples were selected from the market and named Feed-1, Feed-2, Feed-3, and Feed-4 in order. The test feed samples were weighed sequentially, pulverized, and then transferred to sterile water to make the concentration 0.5 g / mL, and shaken uniformly at room temperature to produce a uniform dispersion of the test sample. 50 μL of the sample uniform dispersion was taken and added to 30 mL of the conventional Sabouraud liquid medium, and cultured with shaking at 28 °C and 200 rpm for 24 h, and then the sample was collected to form the test solution of the sample to be discriminated.

[0054] Second step, measurement of the test solution of the sample to be identified: Dissolve the nanobody or monoclonal antibody of AFT-YJFZ01 in a conventional ELISA coating buffer to form a coating solution with a concentration of 0.2 - 8.0 μg / mL, and further add it to a 96-well immunoplate at 200 μL / well, then leave it overnight at 4°C or for 2 hours or more at 37°C. Discard the coating solution in the immunoplate, and further wash the immunoplate with a conventional washing solution for ELISA. Next, use skim milk powder with a concentration of 1% or more as a blocking solution, add 300 μL to each well, leave it at room temperature or block it at 37°C for 1 hour or more, then discard the blocking solution and wash the immunoplate with a conventional washing solution for ELISA. Next, appropriately dilute the test solution with a conventional phosphate buffer with a pH close to neutral, and add 200 μL to each well, or add 200 μL of the solution of the toxin-producing ability index molecule AFT-YJFZ01 of the present invention with stepwise concentrations (the concentrations are 0.00003, 0.0003, 0.003, 0.03, 0.3, 3, 30, 300 ng / mL and are used to obtain a calibration curve) to each well, leave it at room temperature or block it at 37°C for 1 hour or more, then discard the liquid and further wash the immunoplate with a conventional washing solution for ELISA. Next, appropriately dilute the rabbit-derived polyclonal antibody of AFT-YJFZ01 with a conventional phosphate buffer with a pH close to neutral, add 200 μL to each well, leave it at room temperature or block it at 37°C for 1 hour or more, then discard the liquid and wash the immunoplate with a conventional washing solution for ELISA. Next, dilute the commercially available horseradish peroxidase-labeled goat anti-rabbit antibody with a conventional phosphate buffer with a pH close to neutral according to the instructions, add 200 μL to each well, leave it at room temperature or block it at 37°C for 1 hour or more, then discard the liquid and wash the immunoplate with a conventional washing solution for ELISA. Next, add the conventional chromogenic solution and stop solution for ELISA in sequence, and finally read and calculate the AFT-YJFZ01 content with a microplate reader.

[0055] Step 3, Evaluation of Identification Results: When the content of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium in the sample to be identified is high, it indicates that the sample to be identified contains strains with strong toxin-producing ability of aflatoxin. The results of measuring the content of AFT-YJFZ01 in Feed-1, Feed-2, Feed-3, and Feed-4 by the above technical means are that the content of AFT-YJFZ01 per milliliter of the culture solution is 0.6 ng, 22 ng, 13 ng, and 69 ng in sequence. As can be seen from this result, in the Feed-1 sample, the content of AFT-YJFZ01 per milliliter of the culture solution is 1.0 ng or less, and it does not contain strains with strong toxin-producing ability of aflatoxin, and the contamination risk is very low. Taking the addition of 50 μL of the sample uniform dispersion liquid to 30 mL of the medium as an example, in Feed-2, Feed-3, and Feed-4, the content of AFT-YJFZ01 per milliliter of the culture solution is all 10 ng or more, and they contain strains with strong toxin-producing ability of aflatoxin.

[0056] (Appendix) (Appendix 1) The toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium, characterized in that the amino acid sequence of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium is shown by SEQ ID NO: 1.

[0057] (Appendix 2) A method for identifying the toxin-producing ability of aflatoxin toxin-producing bacteria in the system based on the detected content of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium, characterized in that the amino acid sequence of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium is shown by SEQ ID NO: 1.

[0058] (Appendix 3) Use for identifying the aflatoxin-producing ability of Aspergillus aflatoxin-producing strains, (1) providing a nanobody or monoclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium; (2) To provide a polyclonal antibody against the toxin-producing ability index molecule AFT-YJFZ01 of aflatoxin-producing fungi, (3) Preparation of the test solution of the strain to be identified: Culturing the strain to be identified, diluting it to obtain the test solution of the strain to be identified, (4) Measuring the aflatoxin-producing ability of the strain to be identified, including Adopting an indirect non-competitive double antibody sandwich method to identify the aflatoxin-producing ability of Aspergillus aflatoxin-producing strains, Coating the immunosorbent plate with the nanobody or monoclonal antibody of AFT-YJFZ01 and washing the plate, Adding a blocking solution for blocking and washing the plate, Adding the test solution for reaction and washing the plate, Adding the polyclonal antibody of AFT-YJFZ01 for reaction and washing the plate, Adding a horseradish peroxidase-labeled antibody that binds to the specific polyclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of aflatoxin-producing fungi for reaction and washing the plate, Adding a chromogenic solution for reaction, Adding a stop solution, reading and calculating with a microplate reader to obtain the content of the toxin-producing ability index molecule AFT-YJFZ01 of aflatoxin-producing fungi, and the higher the content of the toxin-producing ability index molecule AFT-YJFZ01 of aflatoxin-producing fungi in the test solution of the strain to be identified, the stronger the aflatoxin-producing ability of the aflatoxin-producing strain, including The use is characterized in that the amino acid sequence of the toxin-producing ability index molecule AFT-YJFZ01 of aflatoxin-producing fungi is shown in SEQ ID NO: 1.

[0059] (Appendix 4) The test strain to be identified is cultured in a conventional Czapek medium or a medium suitable for the growth of other strains, the environmental temperature for culturing is 15 to 35°C, the culturing time is 12 h or more, the mixture of the medium and the culture is sufficiently homogenized, and further diluted 1 to 10 times with sterile water to obtain a test solution of the test strain to be identified, which is the step (3) described in Supplementary Note 3.

[0060] (Supplementary Note 5) Step a: A coating solution with a concentration of 0.2 to 8.0 μg / mL is prepared by mixing the nanobody or monoclonal antibody of AFT-YJFZ01 with an ELISA coating buffer, added to an immunoplate, and left overnight at 4°C or left for 2 h or more at 37°C. Then, the coating solution in the immunoplate is discarded, and the immunoplate is washed with a conventional washing solution for ELISA. Next, skim milk powder with a concentration of 1% or more is used as a blocking solution, left at room temperature or blocked for 1 h or more at 37°C, then the blocking solution is discarded, and the immunoplate is washed with a conventional washing solution for ELISA. Step b: Next, the test solution of the test strain to be identified is appropriately diluted with a conventional phosphate buffer with a pH close to neutral, added to the wells of the immunoplate, left at room temperature or blocked for 1 h or more at 37°C, then the liquid is discarded, and the immunoplate is washed with a conventional washing solution for ELISA. Step c: Next, the polyclonal antibody of AFT-YJFZ01 is appropriately diluted with a conventional phosphate buffer with a pH close to neutral, added to the wells of the immunoplate, left at room temperature or blocked for 1 h or more at 37°C, then the liquid is discarded, and the immunoplate is washed with a conventional washing solution for ELISA. Step d: Next, a commercially available horseradish peroxidase-labeled antibody is diluted as needed with a conventional phosphate buffer with a pH close to neutral, added to the wells of the immunoplate, left at room temperature or blocked for 1 h or more at 37°C, then the liquid is discarded, and the immunoplate is washed with a conventional washing solution for ELISA. Step e: Next, add the conventional chromogenic solution and stop solution of ELISA in sequence, and finally read and calculate the result of the AFT-YJFZ01 content by a microplate reader, which is the step (4) described in Appendix 3, characterized by the use described in Appendix 3.

[0061] (Appendix 6) A use for discriminating whether there is a strong aflatoxin toxin-producing strain with toxin-producing ability in a sample, specifically: (1) Providing a nanobody or monoclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of an aflatoxin toxin-producing bacterium; (2) Providing a polyclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of an aflatoxin toxin-producing bacterium; (3) Preparation of a sample to be discriminated: Culturing and diluting the sample to be discriminated to obtain a test solution of the sample to be discriminated; (4) Used for discriminating whether there is a strong aflatoxin toxin-producing strain with toxin-producing ability in a sample, and discriminating the sample by adopting an indirect non-competitive double antibody sandwich method, Coating a nanobody or monoclonal antibody of AFT-YJFZ01 on an immunoplate and washing the plate; Adding a blocking solution for blocking and washing the plate; Adding a sample solution to be discriminated for reaction and washing the plate; Adding a polyclonal antibody of AFT-YJFZ01 for reaction and washing the plate; Adding a horseradish peroxidase-labeled antibody that binds and reacts with the polyclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of an aflatoxin toxin-producing bacterium and washing the plate; Adding a chromogenic solution for reaction; Adding a stop solution, reading and calculating with a microplate reader to obtain the content of the toxin-producing ability index molecule AFT-YJFZ01 of an aflatoxin toxin-producing bacterium, including; (5) Evaluating the discrimination result, The higher the content of the toxin-producing force index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium in the sample to be identified, the stronger the toxin-producing force, which is the aflatoxin-producing ability of the strain to be identified. Based on the calculation results of the microplate reader, the content of the toxin-producing force index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium per unit volume of the sample test solution is obtained, and it is determined whether the sample to be identified contains a strain with strong aflatoxin toxin-producing force. The amino acid sequence of the toxin-producing force index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium is shown in SEQ ID NO: 1.

[0062] (Appendix 7) Weigh the sample to be identified, transfer it to sterile water, shake it uniformly at room temperature to prepare a uniform dispersion of the test sample, add 10 - 1000 μL of the sample uniform dispersion to 6 - 600 mL of the conventional Czapek medium or other media suitable for the growth of aflatoxin toxin-producing bacteria, and shake-culture at 15 - 35 °C at 200 ± 50 rpm. After culturing for 6 - 24 h, sample to form a test solution of the sample to be identified. This is the step (3) as described in Appendix 6, characterized in that the sample to be identified is soil, traditional Chinese medicine materials, agricultural products or feed.

[0063] (Appendix 8) Step a: Mix the nanobody or monoclonal antibody of AFT-YJFZ01 with ELISA coating buffer to prepare a coating solution with a concentration of 0.2 - 8.0 μg / mL, add it to the immunoplate, and leave it at 4 °C overnight or at 37 °C for 2 h or more. Discard the coating solution in the immunoplate, wash the immunoplate with the conventional washing solution for ELISA, then use skim milk powder with a concentration of 1% or more as the blocking solution, leave it at room temperature or block it at 37 °C for 1 h or more, then discard the blocking solution and wash the immunoplate with the conventional washing solution for ELISA. Step b: Next, appropriately dilute the test solution of the sample with a conventional phosphate buffer with a pH close to neutral, add it to the wells of the immunoplate, leave it at room temperature or block it at 37°C for 1 hour or more, then discard the liquid, and wash the immunoplate with a conventional washing solution for ELISA. Step c: Next, appropriately dilute the polyclonal antibody of AFT-YJFZ01 with a conventional phosphate buffer with a pH close to neutral, add it to the wells of the immunoplate, leave it at room temperature or block it at 37°C for 1 hour or more, then discard the liquid, and wash the immunoplate with a conventional washing solution for ELISA. Step d: Next, appropriately dilute a commercially available horseradish peroxidase-labeled antibody with a conventional phosphate buffer with a pH close to neutral as needed, add it to the wells of the immunoplate, leave it at room temperature or block it at 37°C for 1 hour or more, then discard the liquid, and wash the immunoplate with a conventional washing solution for ELISA. Step e: Next, sequentially add a conventional chromogenic solution and a stop solution for ELISA, and finally read and calculate the result of the AFT-YJFZ01 content by a microplate reader, which is the step (4) described in appendix 6.

[0064] (Appendix 9) The use according to appendix 3 or 6, characterized in that the polyclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium is different from the animal source of the nanobody or monoclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium.

[0065] (Appendix 10) The use according to appendix 3 or 6, characterized in that a calibration curve is prepared using a solution of the toxin-producing ability index molecule AFT-YJFZ01 with a stepwise concentration gradient, and the content of AFT-YJFZ01 is obtained using the calibration curve based on the result of the microplate reader.

Claims

1. The amino acid sequence of the toxin-producing ability indicator molecule AFT-YJFZ01 of the aflatoxin-producing bacterium is characterized in that it is represented by SEQ ID NO: 1, and it is the toxin-producing ability indicator molecule AFT-YJFZ01 of the aflatoxin-producing bacterium.

2. Based on the detected content of the toxin-producing ability indicator molecule AFT-YJFZ01 of the aflatoxin-producing bacterium, the toxin-producing ability of the aflatoxin-producing bacterium to be identified is identified, and the amino acid sequence of the toxin-producing ability indicator molecule AFT-YJFZ01 of the aflatoxin-producing bacterium is represented by SEQ ID NO:

1. A method for identifying the toxin-producing ability of the aflatoxin-producing bacterium to be identified, characterized in that.

3. A method for identifying the aflatoxin-producing ability of Aspergillus aflatoxin-producing strains, comprising: (1) providing a nanobody or monoclonal antibody of the toxin-producing ability indicator molecule AFT-YJFZ01 of the aflatoxin-producing bacterium; (2) providing a polyclonal antibody of the toxin-producing ability indicator molecule AFT-YJFZ01 of the aflatoxin-producing bacterium; (3) preparation of a test solution of the strain to be identified: culturing the strain to be identified, diluting it, and obtaining a test solution of the strain to be identified; (4) measuring the aflatoxin-producing ability of the strain to be identified, adopting an indirect non-competitive double antibody sandwich method to identify the aflatoxin-producing ability of Aspergillus aflatoxin-producing strains, coating the immunosorbent plate with a nanobody or monoclonal antibody of AFT-YJFZ01 and washing the plate; adding a blocking solution for blocking and washing the plate; adding the test solution for reaction and washing the plate; adding a polyclonal antibody of AFT-YJFZ01 for reaction and washing the plate; adding a horseradish peroxidase-labeled antibody that binds to the specific polyclonal antibody of the toxin-producing ability indicator molecule AFT-YJFZ01 of the aflatoxin-producing bacterium for reaction and washing the plate; adding a chromogenic solution for reaction. Add a stop solution, read with a microplate reader, calculate, and obtain the content of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin-producing bacterium. The higher the content of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin-producing bacterium in the test solution of the strain to be discriminated, the stronger the aflatoxin-producing ability of the aflatoxin-producing strain. The method is characterized in that the amino acid sequence of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin-producing bacterium is represented by SEQ ID NO:

1.

4. The method according to claim 3, wherein the strain to be discriminated is cultured in a conventional Czapek medium or a medium suitable for the growth of other strains, the environmental temperature of the culture is 15 to 35 ° C, the culture time is 12 h or more, the mixture of the medium and the culture is sufficiently homogenized, and further diluted 1 to 10 times with sterile water to obtain the test solution of the strain to be discriminated in the step (3).

5. Step a: Prepare a coating solution of 0.2 to 8.0 μg / mL by mixing the nanobody or monoclonal antibody of AFT-YJFZ01 with an ELISA coating buffer, add it to an immunoplate, and leave it overnight at 4 ° C or for 2 h or more at 37 ° C. Discard the coating solution in the immunoplate, wash the immunoplate with a conventional washing solution for ELISA, then use skim milk powder with a concentration of 1% or more as a blocking solution, leave it at room temperature or block it at 37 ° C for 1 h or more, then discard the blocking solution, and wash the immunoplate with a conventional washing solution for ELISA. Step b: Next, appropriately dilute the test solution of the strain to be discriminated with a conventional phosphate buffer with a pH close to neutral, add it to the wells of the immunoplate, leave it at room temperature or block it at 37 ° C for 1 h or more, then discard the liquid, and wash the immunoplate with a conventional washing solution for ELISA. Step c: Next, appropriately dilute the polyclonal antibody of AFT-YJFZ01 with a conventional phosphate buffer with a pH close to neutral, add it to the wells of the immunoplate, leave it at room temperature or block it at 37 ° C for 1 h or more, then discard the liquid, and wash the immunoplate with a conventional washing solution for ELISA. Step d: Next, dilute the commercially available horseradish peroxidase-labeled antibody with a conventional phosphate buffer with a pH close to neutral as needed, add it to the wells of the immunoplate, leave it at room temperature or block it at 37 °C for 1 h or more, then discard the liquid and wash the immunoplate with a conventional washing solution for ELISA, Step e: Next, add a conventional chromogenic solution and stop solution for ELISA in sequence, and finally read and calculate the result of the AFT-YJFZ01 content by a microplate reader, which is the step (4) according to claim 3.

6. A method for discriminating whether there is an aflatoxin-producing strain with strong toxin-producing ability in a sample, (1) providing a nanobody or monoclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of an aflatoxin-producing bacterium; (2) providing a polyclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of an aflatoxin-producing bacterium; (3) Preparation of a sample to be discriminated: culturing and diluting the sample to be discriminated to obtain a test solution of the sample to be discriminated; (4) being used to discriminate whether there is an aflatoxin-producing strain with strong toxin-producing ability in the sample, and discriminating the sample by adopting an indirect non-competitive double antibody sandwich method, coating the immunoplate with a nanobody or monoclonal antibody of AFT-YJFZ01 and washing the plate; adding a blocking solution to block and washing the plate; adding the sample solution to be discriminated to react and washing the plate; adding a polyclonal antibody of AFT-YJFZ01 to react and washing the plate; adding a horseradish peroxidase-labeled antibody that binds and reacts with the polyclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of an aflatoxin-producing bacterium and washing the plate; adding a chromogenic solution to react; adding a stop solution, reading with a microplate reader, calculating, and obtaining the content of the toxin-producing ability index molecule AFT-YJFZ01 of an aflatoxin-producing bacterium; (5) evaluating the discrimination result. The higher the content of the toxin-producing force index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium in the sample to be identified, the stronger the toxin-producing force, which is the aflatoxin-producing ability of the strain to be identified. Based on the calculation results of the microplate reader, the content of the toxin-producing force index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium per unit volume of the test sample solution is obtained, and it is determined whether the sample to be identified contains a strain with strong aflatoxin toxin-producing force. The amino acid sequence of the toxin-producing force index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium is shown in SEQ ID NO:

1.

7. Weigh the sample to be identified, transfer it to sterile water, shake it uniformly at room temperature to produce a uniformly dispersed test sample solution, add 10 - 1000 μL of the sample uniformly dispersed solution to 6 - 600 mL of the conventional Czapek medium or other medium suitable for the growth of aflatoxin toxin-producing bacteria, shake and culture at 15 - 35 °C at 200 ± 50 rpm, sample after culturing for 6 - 24 h to form a test solution of the sample to be identified. The method according to claim 6, characterized in that it is the step (3) where the sample to be identified is soil, traditional Chinese medicine materials, agricultural products or feed.

8. Step a: Mix the nanobody or monoclonal antibody of AFT-YJFZ01 with an ELISA coating buffer to prepare a coating solution with a concentration of 0.2 - 8.0 μg / mL, add it to an immunoplate, and leave it at 4 °C overnight or at 37 °C for 2 h or more. Discard the coating solution in the immunoplate, wash the immunoplate with a conventional ELISA washing solution. Next, use skim milk powder with a concentration of 1% or more as a blocking solution, leave it at room temperature or block it at 37 °C for 1 h or more, then discard the blocking solution and wash the immunoplate with a conventional ELISA washing solution. Step b: Next, appropriately dilute the test solution of the sample with a conventional phosphate buffer with a pH close to neutral, add it to the wells of the immunoplate, leave it at room temperature or block it at 37 °C for 1 h or more, then discard the liquid and wash the immunoplate with a conventional ELISA washing solution. Step c: Next, appropriately dilute the polyclonal antibody of AFT-YJFZ01 with a conventional phosphate buffer with a pH close to neutral, add it to the wells of the immunoplate, leave it at room temperature or block it at 37°C for 1 h or more, then discard the liquid, and wash the immunoplate with a conventional washing solution for ELISA. Step d: Next, appropriately dilute a commercially available horseradish peroxidase-labeled antibody with a conventional phosphate buffer with a pH close to neutral as needed, add it to the wells of the immunoplate, leave it at room temperature or block it at 37°C for 1 h or more, then discard the liquid, and wash the immunoplate with a conventional washing solution for ELISA. Step e: Next, sequentially add a conventional chromogenic solution and a stop solution for ELISA, and finally read and calculate the result of the AFT-YJFZ01 content by a microplate reader, which is the step (4) according to claim 6.

9. The method according to claim 3 or 6, characterized in that the polyclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium is different from the animal source of the nanobody or monoclonal antibody of the toxin-producing ability index molecule AFT-YJFZ01 of the aflatoxin toxin-producing bacterium.

10. The method according to claim 3 or 6, characterized in that a calibration curve is prepared using a solution of the toxin-producing ability index molecule AFT-YJFZ01 with a stepwise concentration gradient, and the content of AFT-YJFZ01 is obtained using the calibration curve based on the result of the microplate reader.