CN113721022B - Quick identification method for relative abundance of aflatoxin-producing bacteria in farmland and application thereof - Google Patents

Abstract

The invention provides a method for rapidly identifying relative abundance of aflatoxin toxigenic bacteria in farmland and application thereof. Culturing a soil sample to be detected by using a Chlamydia medium or other medium suitable for the growth of aspergillus flavus with toxicity, and preparing a liquid to be detected of the soil sample to be detected for later use; (2) The indirect non-competitive double antibody sandwich method is adopted to identify and compare the relative abundance of the toxigenic fungi of the aflatoxin in farmland soil; (3) The relative abundance of the toxigenic fungi of the aflatoxin in farmland soil is known by comparing the AFT-YJFZP008 concentration; the molecule for indicating the virulence of aflatoxin-producing bacteria refers to AFT-YJFZP008 peptide, and the amino acid sequence of the molecule is shown in SEQ ID NO. 1. The method is used for identifying the relative abundance of the toxigenic fungi of the aflatoxin in the farmland soil sample, has high identification speed, strong timeliness and practicability, is easy to popularize and apply, and can be applied to monitoring the occurrence risk of the toxigenic fungi of the aflatoxin in the field.

Description

Quick identification method for relative abundance of aflatoxin-producing bacteria in farmland and application thereof

Technical Field

The invention relates to a method for rapidly identifying relative abundance of aflatoxin toxigenic bacteria in farmland and application thereof.

Background

Aflatoxin has strong toxicity and great harm, is the pollutant with the largest variety of polluted foods, generally presents a pollution aggravating trend in recent years, and seriously threatens the food safety and the health of people. Aflatoxin is the most toxic mycotoxin in nature, wherein aflatoxin B1 is a class I carcinogen identified by International cancer research organization (International Agency for Research on Cancer, IARC), and has caused excessive poisoning events of human and animal populations, and becomes one of the main causes of high incidence of liver cancer cases. Statistics of data retrieved according to the last 5 years Web of Science: the aflatoxin pollutes food and raw materials more than 110 kinds, and the high-concentration pollutants are first.

Research has shown that aflatoxin is mainly produced by toxic fungi such as aspergillus flavus. Taking peanuts as an example, the peanuts are provided with aflatoxin-producing strains in the field, and after the peanut is harvested, the strains enter packaging bags, transport vehicles, warehouses, processing lines and the like along with the peanuts, and once the conditions are proper, a large amount of aflatoxins can be produced, so that the consumption safety and the life health safety of people of the peanuts and products thereof are threatened.

In order to reduce the toxic fungi with or without aflatoxin after delivery of peanuts, the international well-known research institutions such as the national institute of semi-arid of the United nations and the national institute of grain and agriculture (FAO) of the United states department of agriculture, and the like, research, prevention and control are carried out on the toxic fungi with aflatoxin as soil-borne pathogens of crops such as peanuts.

However, how does the abundance of the toxigenic fungus of aflatoxin in farmland soil be identified? This is critical to the accuracy and timeliness of the formulation of the prevention and control strategy. The existing reported method for identifying the abundance of the aflatoxin-producing fungi in farmland soil is mainly a colony count method, namely, a soil sample is cultivated in a solid Chlamydia medium or a flat-plate medium with equivalent effect until colonies are grown, and then the colony count in each gram of soil is calculated. In the existing method, the number of colonies growing on the flat plate is too large to be overlapped or too small to be few, which can affect the accuracy of the calculation result, and particularly, the colonies can grow only by culturing the sample for many days, so that the requirement of instantaneity is difficult to meet.

In order to solve the problems, the inventor groups successfully find a molecule indicating the virulence of the aflatoxin-producing bacteria after more than ten years of attack researches, and the first research finds that: the indicator molecules can be produced after the soil is cultured for 6-24 hours, and particularly, the concentration of the indicator molecules and the result of the existing colony count method show positive correlation after the soil sample is cultured for 6-24 hours, so that the method for rapidly identifying the relative abundance of the aflatoxin-producing bacteria in the farmland is invented, the method can be further used for monitoring the risk of the occurrence of the aflatoxin-producing bacteria in the field, and a scientific basis is provided for timely prevention and control and early prevention and early control.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides a method for rapidly identifying the relative abundance of aflatoxin-producing bacteria in farmland and application thereof, which is used for identifying the relative abundance of aflatoxin-producing bacteria in farmland soil samples, has the advantages of high identification speed, strong timeliness and practicality and is easy to popularize and apply.

In order to solve the technical problems, the invention adopts the following technical scheme:

a method for rapidly identifying relative abundance of aflatoxin-producing bacteria in farmland comprises the following steps:

(1) Culturing a soil sample to be detected by using a Chlamydia medium or other culture mediums suitable for the growth of the toxic aspergillus flavus, and preparing a liquid to be detected of the soil sample to be identified for later use;

(2) The method for identifying and comparing the relative abundance of the aflatoxin-producing bacteria in farmland soil by adopting an indirect non-competitive double antibody sandwich method comprises the following steps:

a, adding a liquid to be tested into a hole of an enzyme-labeled plate coated with a nanometer antibody or a monoclonal antibody of a molecule AFT-YJFZP008 for indicating the toxicity of aflatoxin toxigenic bacteria, reacting, and washing the plate;

b, adding a molecular AFT-YJFZP008 polyclonal antibody for indicating the toxicity of aflatoxin toxigenic bacteria for reaction, and washing the plate;

c, adding a horseradish peroxidase labeled antibody which is subjected to a combination reaction with a polyclonal antibody of a molecule AFT-YJFZP008 for indicating the toxicity of aflatoxin-producing bacteria, reacting, and washing a plate;

d, adding a color development liquid for reaction; and adding a stop solution, and reading and calculating a result by using an enzyme label instrument.

The method comprises the following specific steps:

a, adding 100-200 mu L of the liquid to be tested into a hole of an enzyme-labeled plate coated with a nanometer antibody or a monoclonal antibody of a molecule AFT-YJFZP008 for indicating the toxicity of aflatoxin toxin-producing bacteria at the bottom of the hole, standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid after reaction, washing the enzyme-labeled plate,

b, adding AFT-YJFZP008 rabbit-source polyclonal antibody (100-200 mu L is added in each hole), standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding liquid, washing the ELISA plate with the liquid,

c, adding horseradish peroxidase labeled goat anti-rabbit antibody (100-200 mu L per hole), standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding liquid, washing the ELISA plate,

and d, sequentially adding ELISA color development liquid and stop solution, and finally reading and calculating the concentration of AFT-YJFZP008 in the sample to be detected by an enzyme-labeling instrument.

(3) By comparing the AFT-YJFZP008 concentration, the relative abundance of the aflatoxin-producing bacteria in the farmland soil is known, namely the abundance order, namely the determined AFT-YJFZP008 concentration and the relative abundance of the aflatoxin-producing bacteria in the soil are in positive correlation, namely the higher the determined AFT-YJFZP008 concentration is, the higher the relative abundance of the aflatoxin-producing bacteria in the soil is, and the higher the risk of the corresponding farmland-producing bacteria of the soil sample is;

the molecule for indicating the virulence of aflatoxin-producing bacteria refers to AFT-YJFZP008 peptide, and the amino acid sequence of the molecule is shown in SEQ ID NO. 1.

According to the scheme, a series of molecular AFT-YJFZP008 pure product solution (100-200 mu L per hole) which plays a role of a standard substance and indicates the toxicity of aflatoxin toxin-producing bacteria is used for replacing a liquid to be tested, and is used for manufacturing a standard curve, so that the concentration of AFT-YJFZP008 in a sample to be tested is calculated.

According to the scheme, the method comprises the following steps of culturing a soil sample to be detected by using a Chlamydia medium or other medium suitable for the growth of toxic aspergillus flavus and preparing a liquid to be detected of the soil sample to be identified: weighing a soil sample to be detected, transferring the soil sample to a sample diluent, vibrating at room temperature until the soil sample is uniform, preparing a uniform dispersion of the sample to be detected, taking 10-1000 mu L of the uniform dispersion of the sample to be detected, adding the uniform dispersion of the sample to be detected into a culture medium containing 6-600mL of a conventional Soxhlet liquid culture medium or other culture medium suitable for the growth of toxic Aspergillus flavus, placing the culture medium at 28 ℃ for 200rpm for vibrating culture, and sampling after culturing for 6-24 hours to form the sample to be detected of the sample to be identified.

The culture medium is a conventional culture medium, can be self-matched, and can be obtained by directly purchasing commodity.

According to the scheme, the sample diluent is 0.01mol/L phosphate buffer solution containing 0.1% sorbitol and 0.1% soft sugar, and the preparation method comprises the following steps of: sorbitol and soft sugar 0.5g, naCl 4.0g, na ₂ HPO ₄ ·12H ₂ O 1.45g、KCl 0.1g、KH ₂ PO ₄ 0.1g, deionized water was added to a volume of 500mL.

According to the scheme, the hole bottom is coated with a nano antibody or a monoclonal antibody of a molecule AFT-YJFZP008 for indicating the toxicity of aflatoxin-producing bacteria, and the preparation method comprises the following steps: dissolving the AFT-YJFZP008 nano antibody or monoclonal antibody in ELISA coating buffer solution to form 0.2-8.0 mug/mL coating solution, adding the coating solution into an ELISA plate (100-200 mug/hole), standing overnight at 4 ℃ or standing at 37 ℃ for not less than 2 hours, removing the coating solution in the ELISA plate, and washing the ELISA plate with ELISA conventional washing liquid; then adding ELISA routine blocking solution (200-300 mu L is added to each hole), standing at room temperature or 37 ℃ for blocking for at least 1h, discarding the blocking solution, and washing the ELISA plate with ELISA routine washing solution.

According to the scheme, the polyclonal antibody of the molecule AFT-YJFZP008 for indicating the virulence of the aflatoxin-producing bacteria is different from the animal source of the nanometer antibody or the monoclonal antibody of the molecule AFT-YJFZP008 for indicating the virulence of the aflatoxin-producing bacteria, and the molecule for indicating the virulence of the aflatoxin-producing bacteria can be directly used as an antigen to prepare and obtain the nanometer antibody or the monoclonal antibody and the rabbit-source polyclonal antibody, in particular:

the nanometer antibody or monoclonal antibody of the molecule AFT-YJFZP008 for indicating the toxicity of aflatoxin-producing bacteria can be obtained by the following method: AFT-YJFZP008 is used as an immune antigen, alpaca or Balb/c mice are immunized by a conventional mode, and then a known conventional nano antibody or mouse monoclonal antibody preparation technical scheme is utilized to develop and obtain the alpaca or Balb/c mice;

the polyclonal antibody of the molecule AFT-YJFZP008 for indicating the virulence of aflatoxin-producing bacteria can be obtained by the following method: the AFT-YJFZP008 is used as an immune antigen, a conventional mode is adopted to immunize test rabbits such as New Zealand white rabbits, and a known conventional polyclonal antibody preparation technical scheme is utilized to develop and obtain a rabbit-derived polyclonal antibody of the molecular AFT-YJFZP008 for indicating the virulence of aflatoxin-producing bacteria;

the horseradish peroxidase-labeled antibody which is subjected to a combination reaction with the polyclonal antibody of the molecule AFT-YJFZP008 for indicating the toxicity of the aflatoxin-producing strain is a horseradish peroxidase-labeled goat anti-rabbit antibody, and can be directly purchased.

The ELISA coating buffer solution refers to a conventional carbonate buffer solution, and the preparation method comprises the following steps: weighing NaHCO ₃ 1.465g、Na ₂ CO ₃ 0.795g, deionized water is added to fix the volume to 500mL.

The ELISA chromogenic liquid refers to conventional hydrogen peroxide and TMB chromogenic liquid for ELISA.

The stop solution refers to a conventional chromogenic stop solution for ELISA: 2mol/L sulfuric acid aqueous solution, the preparation method is as follows: 44mL of concentrated sulfuric acid is added into 300mL of deionized water, stirred until cooling is achieved, and finally the volume is fixed to 400 mL.

The method for rapidly identifying the relative abundance of the aflatoxin-producing bacteria in the farmland is applied to monitoring the risk of the occurrence of the aflatoxin-producing bacteria in the farmland.

According to the invention, the first found aflatoxin-producing strain virulence indicator molecule AFT-YJFZP008 can be produced after being cultured for 6-24 hours, especially, the indicator molecule has positive correlation with the result of the existing colony count method after being cultured for 6-24 hours in a soil sample, and the nanometer antibody or monoclonal antibody of the aflatoxin-producing strain indicator molecule AFT-YJFZP008 and the rabbit-derived polyclonal antibody are combined, and a sandwich immunodetection method is constructed by the antibodies, so that the method has the advantages of high speed, high timeliness, high practicability and the like, is easy to popularize and apply, provides key grippers and scientific basis for timely finding out the occurrence risk of the aflatoxin producing strain in the farmland and implementing early prevention and early control, and has important significance for promoting the high-quality development of the agricultural industry and guaranteeing the food safety.

The invention has the beneficial effects that:

1. can be used for identifying the relative abundance of the aflatoxin-producing bacteria in farmland soil and timely finding pollution risks,

2. fast-can be completed in 1 day, is easy to operate, has strong practicability, is easy to popularize and apply,

3. has important significance for promoting the high-quality development of agricultural industries such as peanuts and the like and guaranteeing the food safety.

Detailed Description

Example 1 preparation of molecule AFT-YJFZP008 which can function as a Standard substance and indicates virulence of aflatoxin-producing bacteria

The preparation of the culture medium was carried out as follows: 3% (w/v) sucrose, 0.3% (w/v) NaNO ₃ ，0.1％(w/v)K ₂ HPO ₄ ，0.05％(w/v)MgSO ₄ ·7H ₂ O，0.05％(w/v)KCl，0.001％(w/v)FeSO ₄ The culture medium was prepared at pH 6.5. Randomly selecting 10 strains of published publications such as Aspergillus flavus distribution, toxicity and infection research in a China typical peanut production area, namely, a national institute of agriculture, namely, a Shuoshi institute paper, an author Zhang Xing, page 33, namely, published toxigenic strains such as HLJ-1, heNZY-2, huBha-24, JXZS-29-2, LNct-6, GXfc-34, GDZJ-108-19, jcnt-1, huNdx-7, HBHA-8-17 and the like, respectively inoculating the 10 strains into the Boehmeria nivea culture medium, culturing for 5 days at 28 ℃ at 200rpm/min, fully homogenizing and crushing cells by a conventional method, and purifying and obtaining the molecular AFT-YJFZP008 indicating the toxicity of the aflatoxin toxigenic bacteria by using a conventional protein purification system, protein electrophoresis, immunoaffinity and other methods. Test results show that AFT-YJFZP008 can be prepared in the culture of the toxigenic strain, under the same culture conditions, the quantity of AFT-YJFZP008 prepared by HBHA-8-17 is the largest, and the quantity of AFT-YJFZP008 prepared by HLJ-1 is the smallest.

The immunoaffinity method is characterized in that a sample solution is used for diluting an aflatoxin toxigenic bacteria cell disruption solution, filtering is carried out through filter paper, then the solution is continuously added into an immunoaffinity column, the immunoaffinity column is washed by conventional leachates when the solution is basically completely discharged, finally, glycine buffer solution with the pH value of 2.2 or 70% methanol aqueous solution is used for eluting, the solution is timely removed through a conventional ultrafiltration centrifugation method after the eluent is collected, and then, the protein remained in the ultrafiltration centrifuge tube is dissolved out of the ultrafiltration centrifuge tube through sterile water, so that a molecular AFT-YJFZP008 aqueous solution for indicating the toxicity of the aflatoxin toxigenic bacteria can be obtained.

Initial acquisition of the molecule AFT-YJFZP008, which indicates the virulence of the aflatoxin-producing bacteria, was developed by the following method:

(1) Taking an aspergillus flavus strong virulence strain, and culturing to obtain a strain culture and extracellular secretion protein mixture; then breaking the cells of the strain culture to obtain an intracellular protein mixture; combining the extracellular secretion protein mixture and the intracellular protein mixture, and adding carbodiimide for coupling to obtain an aspergillus flavus antigen;

(2) Immunizing a test animal with the aspergillus flavus antigen to obtain a nano antibody library or a monoclonal antibody library;

(3) Obtaining protein combined solutions of aspergillus flavus strains with different virulence, detecting the proteins of the aspergillus flavus strains with different virulence by using the antibodies in the antibody library obtained in the step (2), and obtaining a series of detection signals;

(4) Finding out a nano antibody with a detection signal positively correlated with the aspergillus flavus strain virulence, namely an aspergillus flavus strain virulence indicator molecule antibody, and a protein corresponding to the aspergillus flavus strain virulence indicator molecule antibody, namely an discovered aspergillus flavus strain virulence indicator molecule.

In the scheme, the aspergillus flavus strong virulence strain in the step (1) is separated and identified from the natural world by a conventional method or is obtained by artificial transformation, and the virulence is identified to be not less than 10 mug/kg by a NY/T2311-2013 standard method.

And (3) the aspergillus flavus strains with different virulence in the step (3) are not less than 3 strains, and the virulence is at least 3 layers higher, middle and lower as the result of the identification by the NY/T2311-2013 standard method.

The culture medium adopted in the culture of the aspergillus flavus strong virulence strain is a Chlamydia medium or other nutrients for the normal growth of the aspergillus flavus, the culture time is not less than 12 hours, and the culture environment temperature is 15-35 ℃.

The cell disruption of the strain culture is carried out by a conventional liquid nitrogen grinding method or a cell disruption instrument method.

The amount of the carbodiimide added to the combined extracellular secreted protein mixture and intracellular protein mixture is 0.005-0.1 g per 1.0 mL.

The coupling reaction is carried out at 15-37 ℃ for 2-6 h and at 4-10 ℃ overnight.

The immunization is a conventional immunization mode, and Aspergillus flavus antigens are inoculated. The test animal refers to a white mouse or alpaca or other test animals with similar effects.

According to the scheme, the antibody preparation process refers to a conventional nanobody preparation process or a conventional hybridoma monoclonal antibody preparation process based on cell fusion.

According to the scheme, the detection of the proteins of the aspergillus flavus strains with different virulence is realized by adopting a conventional Western Blot technical process, namely, the proteins of the aspergillus flavus strains with different virulence are transferred onto a nitrocellulose membrane, and then the antibodies in the antibody library are used for detection by a direct method or an indirect method, or other technical processes with similar effects are adopted.

According to the scheme, the direct method refers to coupling the antibodies in the antibody library with a signal material by a conventional method, and then performing an immune binding reaction with the corresponding proteins transferred onto the nitrocellulose membrane.

According to the scheme, the indirect method is that the antibodies in the antibody library are subjected to immune binding reaction with the corresponding proteins transferred onto the nitrocellulose membrane, and then the second antibodies and the conjugate of the signal material are subjected to immune binding reaction with the antibodies bound onto the nitrocellulose membrane.

The signal material in the detection is horseradish peroxidase, colloidal gold, fluorescent material or other materials with similar effects. The detection signal is a chromogenic reaction signal or a spot signal or a fluorescent signal.

After the molecular AFT-YJFZP008 antibody for indicating the toxicity of the aflatoxin-producing bacteria acquires the whole sequence of the aflatoxin early-warning molecule AFT-YJFZP008, the whole peptide fragment or part of the peptide fragment is prepared by using the conventional antibody preparation technical flow.

Example 2 nanobody preparation of molecule AFT-YJFZP008 indicating virulence of Aflatoxin toxigenic bacteria

AFT-YJFZP008 is used as an immune antigen, alpaca or Balb/c mice are immunized by a conventional mode, and then the preparation technical scheme of known conventional nano antibodies or mouse monoclonal antibodies is utilized to develop and obtain the alpaca or Balb/c mice.

Dissolving AFT-YJFZP008 obtained by the preparation method in a conventional PBS buffer solution or normal saline until the concentration is not lower than 0.1mg/mL, mixing and emulsifying with Freund's complete adjuvant in an equal volume, immunizing alpaca by subcutaneous or intradermal multipoint injection at back, and then enhancing immunity for 1 time every 2-4 weeks, wherein Freund's complete adjuvant is replaced by Freund's incomplete adjuvant during enhancing immunity. The immune effect is monitored by adopting a conventional ELISA flow until serum titer of alpaca is not increased any more, then the operations of venous blood collection, total RNA extraction, cDNA synthesis, VHH gene amplification, VHH gene fragment recovery, connection of the VHH gene and a double enzyme digestion pCANTAB 5E (his) carrier, electric conversion of a connection product, construction of a nanobody gene library, rescue of the nanobody gene library and the like of the alpaca are completed according to the method of a patent document CN103866401A, and finally the rescued nanobody gene library is obtained.

Fixing AFT-YJFZP008 obtained by the preparation on solid-phase carriers such as 96-hole ELISA plates according to gradients of 8 mug/hole, 2 mug/hole, 0.5 mug/hole and 0.1 mug/hole, panning the saved nanobody gene library for 2-4 times according to a method of patent document CN103866401A, identifying antibodies generated by each phage clone by using AFT-YJFZP008 and indirect non-competitive ELISA, identifying phage corresponding to positive results as phage positive clones, preparing the nanobody by the positive clones in a conventional mode of nanobody preparation, namely the nanobody of AFT-YJFZP008, for further application research work, preferably characterizing the nanobody with strong specificity and high affinity by ELISA method.

Example 3 preparation of monoclonal antibody against molecule AFT-YJFZP008 indicating virulence of aflatoxin-producing bacteria

AFT-YJFZP008 is used as an immune antigen, alpaca or Balb/c mice are immunized by a conventional mode, and then the preparation technical scheme of known conventional nano antibodies or mouse monoclonal antibodies is utilized to develop and obtain the alpaca or Balb/c mice.

The AFT-YJFZP008 obtained by the preparation is dissolved in a conventional PBS buffer solution or normal saline until the concentration is not lower than 0.1mg/mL, and is mixed and emulsified with Freund's complete adjuvant in an equal volume, BALB/c mice are subjected to subcutaneous or intradermal multipoint injection mode at the back, and then are subjected to boosting for 1 time every 2-4 weeks, and Freund's complete adjuvant is replaced by Freund's incomplete adjuvant during boosting. And (3) monitoring the immune effect by adopting a conventional ELISA flow, after the serum titer of the BALB/c mice is no longer increased, then separating immune mouse spleen cells, fusing the spleen cells with mouse myeloma cells SP2/0, completing the selective culture operation of a semisolid culture medium on hybridoma cells according to a method of patent document CN103849604A, and after a needle point white spot grows on the semisolid culture medium, respectively picking the white spots into 96-hole culture plates with the conventional culture medium of the built-in hybridoma, thereby obtaining the monoclonal hybridoma resource library.

The monoclonal antibody, namely monoclonal antibody, is obtained according to the method of patent document CN103849604A, AFT-YJFZP008 obtained by the preparation is fixed on solid phase carriers such as 96-well ELISA plates according to the gradient of 8 mug/well, 2 mug/well, 0.5 mug/well and 0.1 mug/well, each monoclonal antibody is identified by using an indirect non-competitive ELISA program, positive clones are picked up, and the AFT-YJFZP008 monoclonal antibody is obtained and used for further application research work, and the AFT-YJFZP008 monoclonal antibody with the characteristics of strong specificity and high affinity is preferably detected.

Example 4 preparation of Rabbit-derived polyclonal antibody of molecule AFT-YJFZP008 indicating virulence of aflatoxin-producing bacteria

AFT-YJFZP008 is used as an immune antigen, test rabbits such as New Zealand white rabbits are immunized by a conventional mode, and a known conventional rabbit polyclonal antibody preparation technical scheme is utilized to develop and obtain the antibody.

The prepared molecule AFT-YJFZP008 which indicates the toxicity of aflatoxin-producing bacteria is directly used as an antigen, the solution with the concentration of not less than 0.1mg/mL is mixed and emulsified with Freund's complete adjuvant in an equal volume, new Zealand white rabbits are subjected to subcutaneous or intradermal multipoint injection at the back, then the immunization is enhanced for 1 time every 2-4 weeks, and Freund's complete adjuvant is replaced by Freund's incomplete adjuvant during the enhancement. And (3) monitoring the immune effect by adopting a conventional ELISA flow, and preparing and obtaining serum of the immune animal by a conventional method after the serum titer of the immune animal is not increased, namely the rabbit-derived polyclonal antibody of the molecule AFT-YJFZP008 for indicating the virulence of aflatoxin-producing bacteria.

Example 5 establishment of an immunoassay Rapid detection method for molecular AFT-YJFZP008 indicating virulence of aflatoxin-producing bacteria

Basic operation procedure of AFT-YJFZP008 immune rapid detection method, namely double-antibody sandwich indirect non-competitive ELISA method: coating AFT-YJFZP008 nano antibody in the ELISA plate, and washing the plate; adding a sealing liquid for sealing and washing the plate; adding AFT-YJFZP008 or a liquid to be tested for reaction, and washing the plate; adding AFT-YJFZP008 rabbit-source polyclonal antibody for reaction, and washing the plate; adding horseradish peroxidase labeled goat anti-rabbit antibody for reaction, and washing the plate; adding a color development liquid for reaction; and adding a stop solution, and reading and calculating a result by using an enzyme label instrument. The following work is accomplished using this basic procedure.

Determination of antibody concentration: a plurality of parallel experiments are simultaneously carried out by adopting a chessboard titration method, the nano-antibody coating with different concentrations is adopted, in addition, the rabbit polyclonal antibody is set to be different concentrations, and finally, one proper working concentration of the nano-antibody and the rabbit polyclonal antibody is determined according to the result, namely, the two antibody concentrations corresponding to the point with the OD450nm value of approximately 1.0 are selected under the principle of saving the antibody consumption, ELISA result research shows that the other concentrations of the nano-antibody and the rabbit polyclonal antibody can also be detected, the proper coating concentration of the nano-antibody is 1.6 mug/mL, and the proper concentration of the rabbit polyclonal antibody is 2.0 mug/mL.

Determination of optimal antibody coating conditions: coating is the first step in ELISA method research, and the quality of the coating effect has a very critical effect on the ELISA result. In order to determine the influence of different coating conditions on the detection result, three different conditions of coating at 4 ℃ overnight, constant temperature coating at 37 ℃ for 2h and constant temperature coating at 37 ℃ for 1h are selected to be coated in the hole, and the detection result shows that the three coating modes can be used for coating, and the coating at 4 ℃ is the optimal coating condition.

Determination of optimal blocking agent: after the antibody is coated, in order to avoid interference of unoccupied sites of the ELISA plate hole on subsequent steps of ELISA, inert proteins, namely blocking agents, need to be used for occupying the sites, and improper blocking agents can be combined with the secondary antibody in a non-specific way, so that false positive conditions are caused. The study adopts three different blocking agents of 3% BSA/PBST, 3% skimmed milk powder/PBST and 5% skimmed milk powder/PBST for blocking, and the study results show that although the three blocking agents can achieve the purpose of blocking in different degrees, the blocking effect of 5% skimmed milk powder/PBST is optimal, and the blocking agent is the optimal blocking agent.

Determination of the closing time: the study sets up three kinds of different closure duration of constant temperature closure 1h, 2h, 3h respectively and seals, and the result of detection shows that the numerical value of positive hole OD450nm value/negative hole OD450nm value is the biggest under the setting of 37 ℃ and closure 2h, and positive sample OD450nm value is > 1.0, so that constant temperature closure 2h at 37 ℃ is the best closure time.

Determination of the reaction time of the rabbit polyclonal antibody: the study adopts three reaction durations of 30min, 50min and 1h of constant temperature reaction at 37 ℃ to carry out reaction, and the detection result shows that: the optimal reaction time of the rabbit polyclonal antibody is obtained by reacting for 50min at the constant temperature of 37 ℃.

ELISA standard curve of the molecule AFT-YJFZP008 for indicating the toxicity of aflatoxin-producing bacteria is drawn: AFT-YJFZP008 molecules were diluted to 0.00003, 0.0003, 0.003, 0.03, 0.3, 3, 30, 300ng/mL, 200. Mu.L of each well was used for the inlet, and ELISA standard curves of AFT-YJFZP008 were drawn using the above-described optimum conditions. The correlation coefficient of the double-antibody sandwich ELISA method established under the optimal condition reaches 0.99, and the detection limit of AFT-YJFZP008 molecules reaches 0.05ng/mL, which shows that the detection method has good detection sensitivity and accuracy.

Method specificity evaluation: in order to evaluate the specificity of the above-mentioned immunodetection method of molecular AFT-YJFZP008 for indicating the toxicity of aflatoxin-producing bacteria, several strains of fungi including fusarium oxysporum, aspergillus niger, aspergillus ochraceus, fusarium moniliforme and the like which have certain homology with the aflatoxin-producing bacteria are researched and selected, and the results are shown in table 1 by detecting cell disruption liquid of the fungi cultures, the above-mentioned method has no obvious cross reaction on proteins of fungi with homology with the aflatoxin-producing bacteria, and the established immunorapid detection method of molecular AFT-YJFZP008 for indicating the toxicity of aflatoxin-producing bacteria has good specificity.

TABLE 1 molecular immune rapid detection method for indicating the virulence of aflatoxin toxigenic bacteria

Method repeatability evaluation: in order to evaluate the repeatability of the established molecular AFT-YJFZP008 immune rapid detection method for indicating the virulence of aflatoxin, randomly taking the study of positive 1, positive 2, positive 3, positive 4 and non-virulent strains of 1 aflatoxin, namely negative 5, of 4 strains of aflatoxin, and analyzing the data variation in and among plates of the measurement result. The results of the above study are shown in Table 2, and the calculated intra-plate variation coefficient is 0.6% -5.7%, and the calculated inter-plate variation coefficient is 1.2% -9.1%, which are all below 10%, so that the above method has good repeatability.

TABLE 2 repetitive assay results of AFT-YJFZP008 immune rapid detection method for virulence indicator

Evaluation of accuracy of the method: in order to evaluate the detection accuracy of the method, and also to examine the practicability of the method, peanut and corn samples are selected as examples for research and evaluation, and a molecule AFT-YJFZP008 which indicates the virulence of aflatoxin-producing bacteria is added into the corn and peanut samples, so that the recovery rate of the detection result is improved. The closer the recovery rate is to 100%, the more accurate and practical the method is to be explained. The research results are shown in Table 3, and the results show that the detection method established above is used for detecting the virulence indicator molecule AFT-YJFZP008 in peanuts and corns, and the recovery rate reaches 83% -106%, which shows that the method has high accuracy, can be applied to actual sample detection, and has good practicability.

TABLE 3 molecular immune rapid detection method for indicating the virulence of aflatoxin toxigenic bacteria adding recovery test results

Example 6 quantitative correlation between the toxigenic fungal toxigenic indicator molecules of aflatoxins and the abundance of toxigenic fungi of aflatoxins in soil

6 parts of farmland soil with different pollution levels of peanut aflatoxin are selected, and the method is used for measuring the soil by the established detection method and specifically comprises the following steps.

Firstly, preparing a sample to be identified and a liquid to be tested: sequentially weighing farmland soil samples to be measured, uniformly crushing, transferring the samples into sample diluent, and vibrating the samples at room temperature until the concentration is 0.5g/mL, so as to prepare uniform dispersion liquid of the samples to be measured. And taking 50 mu L of the uniform dispersion liquid of the sample to be detected, adding the uniform dispersion liquid into 30mL of the conventional Nahnsonian culture medium, placing the culture medium at 28 ℃ for 200rpm for shake culture, and sampling after 20h of culture to form the sample to be detected of the sample to be identified.

Step two, the determination of the sample to be identified is carried out: adding 100-200 mu L of the liquid to be tested into the enzyme-labeled plate hole coated with the nanometer antibody of the molecular AFT-YJFZP008 for indicating the toxicity of aflatoxin, or adding 100-200 mu L of the solution of the toxicity indicating molecule AFT-YJFZP008 with the series of concentrations into each hole, standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding the reacted liquid, washing the enzyme-labeled plate, adding 100-200 mu L of the AFT-YJFZP008 rabbit-derived polyclonal antibody into each hole, standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid, washing the enzyme-labeled plate, adding 100-200 mu L of the horseradish peroxidase-labeled goat anti-rabbit antibody into each hole, standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid, washing the enzyme-labeled plate, then sequentially adding the conventional color development liquid and termination liquid for ELISA, and finally reading and calculating the concentration of AFT-YJFZP008 in a farmland soil sample to be tested by an enzyme-labeled instrument.

The abundance of the toxigenic fungi of the aflatoxin in the soil is measured by adopting a conventional colony counting method, and the abundance is sequentially as follows: 278cfu/g, 216cfu/g, 169cfu/g, 155cfu/g, 112cfu/g, 17cfu/g. The measurement results show that: the concentration of the toxicity indicating molecules AFT-YJFZP008 in the 6 soil samples is as follows: 17.2ng/g, 9.6ng/g, 2.0ng/g, 0.9ng/g, 0.2ng/g, 0ng/g, the results indicate that: the higher the concentration of the toxicity indicating molecule AFT-YJFZP008 in the soil, the higher the abundance of the toxic fungus population of aflatoxin in the soil, and the greater the risk of pollution of crops such as peanuts and the like after the production by the aflatoxin.

Example 7 virulence indicator molecule immunization Using A toxigenic fungus of aflatoxin method for identifying and comparing abundance of toxigenic fungi of aflatoxin in farmland soil by rapid detection method

Firstly, preparing a sample to be identified and a liquid to be tested: the peanut flowering period rhizosphere soil samples such as Jilin, liaoning, jiangxi, fujian and the like are selected for 4 parts, and are named as soil sample-1, soil sample-2, soil sample-3 and soil sample-4 in sequence. Sequentially weighing farmland soil samples to be measured, uniformly crushing, transferring the farmland soil samples to be measured into sample diluent of the kit, and vibrating the farmland soil samples to be measured to be uniform at room temperature to obtain uniform dispersion liquid of the samples to be measured, wherein the concentration of the sample diluent is 0.5 g/mL. Taking 10-1000 mu L of the uniform dispersion liquid of the sample to be detected, adding the uniform dispersion liquid into 6-600mL of the Nahnia culture medium of the kit, placing the mixture at 28 ℃ for shake culture at 200rpm, and sampling after culturing for 24 hours to form the sample to be detected of the sample to be identified.

Step two, the determination of the sample to be identified is carried out: adding 100-200 mu L of the solution to be tested into an enzyme-labeled plate hole of the kit, or adding 100-200 mu L of the solution of the virulence indicator molecule AFT-YJFZP008 of the invention with serial concentration into the kit, standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding the reacted liquid, washing the enzyme-labeled plate, adding the AFT-YJFZP008 rabbit-source polyclonal antibody into the kit, adding 100-200 mu L of the solution into each hole, standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid, washing the enzyme-labeled plate, adding 100-200 mu L of the horseradish peroxidase labeled goat anti-rabbit antibody into each hole of the kit, standing at room temperature or 37 ℃ for reaction for not less than 1h, discarding the liquid, washing the enzyme-labeled plate, then sequentially adding ELISA color-developing solution and stop solution into the kit, and finally reading and calculating the concentration of AFT-YJFZP008 in the sample to be tested through an enzyme-labeled instrument.

Thirdly, judging the identification result: if the concentration of the molecule AFT-YJFZP008 which indicates the aflatoxin toxigenic bacteria toxigenic in the sample to be identified is high, the strong toxigenic strain containing the aflatoxin in the sample to be identified is indicated. According to the kit and the application technical scheme thereof, the concentration results of AFT-YJFZP008 in the soil sample-1, the soil sample-2, the soil sample-3 and the soil sample-4 are sequentially: 0.2ng/mL, 0.1ng/mL, 9.6ng/mL, 13.3ng/mL. The result shows that the abundance of the toxin-producing fungi of the aflatoxin in the four farmland soil samples is sequentially from low to high: the concentration of AFT-YJFZP008 in the soil sample-2, the soil sample-1, the soil sample-3 and the soil sample-4 is higher, which indicates that the abundance of the aflatoxin-producing fungi in the soil is higher, and the risk of aflatoxin pollution to crop products such as farm post-partum peanuts and the like corresponding to the soil sample-3 and the soil sample-4 is larger if no effective prevention and control measures are taken.

By combining the specific embodiments, the technical scheme of the invention can be used for identifying the relative abundance of the aflatoxin-producing fungi in farmland soil and finding pollution risks in time, has the advantages of high speed, easy operation, strong practicability and the like, is easy to popularize and apply, has wide popularization and application prospect, and has important significance in promoting the high-quality development of agricultural industries such as peanuts and guaranteeing the food safety.

< 110 > institute of oil crop and oil crop of national academy of agricultural sciences

Quick identification method for relative abundance of aflatoxin toxigenic bacteria in farmland less than 120 and application thereof

＜160＞ 1

＜210＞ 1

＜211＞ 18499

＜212＞ PRT

< 213 > Aspergillus flavus

＜400＞ 1

ALAALASERL EPRALASERP HESERGLYPH ELYSALAPHE ASPSERSERP HEPRLEPRLY 60

SALAPHEPRA SNALAPRASP LYSALAGLYV ALILEPRGLS ERLEHISGLN ASPTHRVALG 120

LYTHRPHEGL YLYSALAILE HISASPGLVA LSERPRVALG LYASPTHRAS PALALELEGL 180

ARGALAILEA SNASPTYRIL EASPSERGLN LEASPLYSAL ALELEPHEGL YALAALAGLY 240

SERALAGLAS PPRVALVALV ALLYSALALE THRASNGLYA LAGLYALAIL ELYSALALEV 300

ALSERHISAS PGLYTHRPHE VALALAASPA LALYSALAAS NASNTYRCYS SERASNGLNV 360

ALGLGLYPRT YRSERLETYR SERGLYARGA LAPRVALVAL GLNTYRALAL EASNARGALA 420

SERMETVALT RPGLGLALAG LNGLNVALSE RGLYLYSALA SERPRSERTY RLETHRALAT 480

HRPRARGALA VALGLGLNSE RLEASPALAI LEARGALAVA LGLYGLNALA THRGLARGAL 540

AVALILETHR ASPILEVALA SNGLNGLNAR GALAVALSER PRSERPHEGL ASPVALTRPS 600

ERGLNPRARG ALATYRGLNG LYTYRPHEHI SSERASNASP ASPLELEASN ARGCYSASPV 660

ALALATHRTH RASPVALTYR TYRSERGLYL YSCYSVALTH RALAPRSERG LYPRCYSGLY 720

GLNLYSCYST YRALALEVAL ASNHISGLPH ESERARGASP ALAASPALAC YSASNGLYGL 780

YGLYILEGLT YRASPSERPR ALAASPTHRP RLEGLPHELY SASPALAGLY SERPRLYSPR 840

VALVALGLNI LEGLYHISGL GLYASPVALG LYVALALAGL ILEGLNASNM ETARGASPAL 900

AGLYTYRGLT HRSERILETH RASPTYRTRP GLYARGASPA LAVALTYRAL ALEASPALAI 960

LETYRGLYIL EASPALAARG ASPGLARGAL ASERLEASPV ALGLPRSERL EPRTRPPRAS 1020

NASPGLYILE PHEARGTRPA RGASPPHEPH EASNHISVAL THRILELYSA SPPHEGLYTR 1080

PASPSERALA PHEASPGLNL YSGLYASNSE RLEGLYLECY SLEPRTHRAS PPHELYSASP 1140

PHEPRCYSAS PVALGLNARG PRPRASPLEA LAALAASNAS PALALYSASP PHETHRASPI 1200

LETHRALAGL YSERSERILE GLYCYSASPG LYVALASNPR GLNTHRGLYL YSASPGLYAL 1260

AGLYGLNMET PHEILEPRLE ASNPRASNAL ATYRSERPRA SNTHRLEASN LYSASPGLYA 1320

SPLEVALTHR GLNGLNASNG LLEGLNGLYL YSASPLEASP GLNASPILEG LLYSLYSASP 1380

LEASNASPGL YGLYSERSER VALGLYVALG LNASNARGLY SASPLEASNP RASNGLYSER 1440

GLNPHEILET HRPRGLYGLY LYSASPLEPR TRPTYRGLNC YSASNGLNGL ILEHISTHRL 1500

EVALSERGLY LELEARGARG ASPASNILEL EPRGLASNLE ASPASPGLYL EPRSERGLNP 1560

HEVALTYRGL LYSASPASNT HRCYSASNAL APRILEPRVA LSERPHEPRV ALALAPRTHR 1620

ASPTHRLYSA SPPRPHELYS ALAILEILET HRLESERALA ARGLEASPTH RPHEALATHR 1680

ILEASNTHRL EPHELYSASP PRPRILEASN METGLYPRIL EPRALATHRT HRASPLETHR 1740

ASNMETASPA RGARGASPPR TYRMETPHEH ISGLNALAAS NLEARGASPG LNCYSASNTY 1800

RSERLEGLNT YRTHRILEGL YASNLYSASP GLNGLLYSAR GGLNARGASP GLNILEILEG 1860

LCYSARGASP THRLEVALIL EPRPRGLYSE RARGASPTHR SERLECYSPR METALAPRPR 1920

ASNSERPHEM ETSERTHRLE PRMETTHRAL AASPPHEARG ASPTHRTHRG LYPHEILEGL 1980

THRASPPRLE LYSASPVALH ISGLYPHEAL ATHRARGASP VALVALGLAL APHEARGASP 2040

TYRALACYSP RTRPASNGLY GLYGLGLVAL SERLELYSGL ALAALASERA LAALALEALA 2100

ALAGLYTYRL YSGLALAGLY LEVALPRPHE GLNVALSERP RTHRTHRLYS GLGLTYRASP 2160

GLGLYLEARG METVALASNL YSGLYLEALA LYSGLGLYAS NGLSERVALG LNVALPRARG 2220

ASNHISALAL ESERSERASP ARGGLHISHI SGLLEALAIL EALASERLYS GLILEASNGL 2280

NILEGLNARG GLILESERPH EASNGLNALA TRPLEARGGL LESERALATH RVALMETASP 2340

HISLELESER GLNARGGLLE VALLEVALLE GLYARGGLPR GLYALAGLGL YVALCYSGLT 2400

HRTHRPRGLY VALLYSGLPR GLYILECYSG LTHRTHRPRG LYVALLYSGL ARGGLLEASP 2460

SERARGGLSE RGLPHEPHEI LEARGPHEAL ALESERTHRT RPALAARGPH EALAASNGLN 2520

METPRASNGL YCYSGLNASP LEILESERTH RCYSLYSPHE ALASERASPA SPALACYSGL 2580

GLLYSPHEGL GLILEALAPR TYRVALASNG LYLYSARGPH EGLGLYTYRL EPRASPALAA 2640

RGPHEGLASN SERASNVALL YSPHEGLASN SERASNVALL YSSERSERVA LVALARGPHE 2700

GLYLYSPRVA LGLYALAVAL GLYSERALAA LATHRALALE LYSPHEGLYT RPTRPSERAL 2760

AASPGLYALA TRPPRGLYAL ALEASPASPP HEVALVALTR PVALGLNLYS LYSPHEHISA 2820

SPSERSERAS NASPSERGLY ASNARGPHEH ISVALLETHR ALAGLNLESE RPHEPRARGP 2880

HELEASPGLA LALETHRTYR PRPRPRLYSP HEASNSERLE ALAASPARGP HEGLNTYRPR 2940

GLYASPLEPH EASPGLNGLY THRTHRILEA RGPHESERSE RCYSSERGLY THRARGPHES 3000

ERTHRVALAL AGLYSERARG PHESERVALA LAGLILELEP RGLYALALYS PHEVALGLYG 3060

LYALASERTH RASPALAPHE ALAASPPRLY SPHEVALTHR ASPASNGLYA SPSERLYSGL 3120

YALAASPARG GLYARGGLYA SPALAGLYSE RPRVALPHES ERPRASPSER LYSGLYPHEP 3180

HETHRALAPR GLYARGGLYG LYGLYGLYGL YTHRPHEGLY VALVALMETG LSERTHRHIS 3240

ARGGLYGLYS ERGLYALALE GLYLEALAPH ESERGLALAL YSGLYILEAS PVALALALYS 3300

PRTHRGLYAR GGLYILEGLN ILEASNASPP RSERILEASN ASPASPSERV ALMETILETY 3360

RALAPRALAV ALARGGLYLE ARGASNSERG LYVALHISGL YTHRPHESER SERARGPRGL 3420

GLNGLGLILE GLNLYSGLYL ETYRALAGLY HISARGGLYP RLEASNGLGL YGLYLETYRA 3480

LAGLARGGLY GLNTHRPRLE PRILELEVAL ALAASPGLYA RGGLYSERAS PCYSSERTHR 3540

THRALAGLYG LYCYSCYSGL YGLYTHRGLY CYSGLNPRAS NGLTHRLEVA LPHEGLYSER 3600

SERASPLEAL AARGGLYVAL ASPPHETHRG LASPPRLELE GLNGLYARGG LYVALGLYSE 3660

RASPALATRP THRVALSERG LSERGLYARG GLYVALLEAR GPRVALSERT HRGLYSERAR 3720

GGLYVALTYR ASPILEARGG LYTYRLYSPR SERALASERS ERGLYSERLY SGLYTYRPRT 3780

HRSERGLNGL NASNTRPVAL GLYTHRLELE LEPRARGHIS ALAGLYGLNC YSGLYGLTYR 3840

HISGLASNLY SHISPHEGLN LEILEASNTH RALAALATYR TRPLYSHISP HETHRSERLE 3900

GLGLLYSHIS PHEVALASPT HRPHEGLYLE HISGLYHISL YSHISGLYGL YPRASNPHEG 3960

LGLNLEPRIL EASNGLNPRA RGHISLEPHE GLYLEHISAR GHISTHRASP TYRSERSERG 4020

LNGLSERTHR SERTYRLYSH ISVALASPGL YPHEGLYILE HISTHRPHEA RGHISVALTY 4080

RASPALAVAL GLNASPLYSI LEALAPHEAL ASERTYRLEG LGLTYRALAA RGILEALAPR 4140

GLNPHEGLYA SPLELYSILE ALASERLEAS NASPSERTYR GLTHRLELYS LYSARGILEP 4200

HEARGLELEG LYTHRPRASP GLASPSERTR PPRGLYVALT HRSERPHEPR ASPTYRLYSI 4260

LELEPHEASP SERASNASNV ALALATHRGL YVALGLNVAL SERTHRGLYG LYTHRPHEGL 4320

YTHRARGILE ASNPRHISGL LESERILEAR GASPPRASPP HETYRASNGL ILETYRVALT 4380

HRGLSERLYS ARGILEGLNA LAPHEVALIL ETYRPRGLAS NPHEASPLYS ILESERALAT 4440

YRVALGLGLY SERSERARGI LESERHISHI SALAGLNTHR LELEGLGLYL EGLYTHRHIS 4500

ARGTYRLEGL SERCYSTHRG LARGILESER TYRLYSGLPR GLYILECYSG LTHRTHRPRG 4560

LYVALLYSIL EVALPRGLGL TYRVALPRIL ETHRLYSILE VALGLNVALG LYASPLEARG 4620

ILEVALTHRP RASPGLYLYS ILETYRASPS ERILETYRVA LARGLYSALA LYSGLSERSE 4680

RSERGLSERS ERASPSERSE RGLSERGLSE RGLSERGLSE RGLASPGLLY SLYSPHETHR 4740

ASPTHRPRVA LLETYRGLYP RLYSLYSMET GLASPASPLE ARGLYSPRLE GLYTHRGLYT 4800

HRASPLETRP PRLYSLYSTH RLELEPHETY RALASERSER HISGLALAIL ESERPHEASP 4860

SERCYSARGL YSTHRSERSE RSERTHRALA THRSERTHRS ERTHRSERTH RGLYALAALA 4920

ALALEPRTHR ALAALAPHEG LYALAVALGL GLYGLYLEME TLEGLYVALV ALLEGLYVAL 4980

LEGLYLELEA SPARGPRPRV ALILEPRLEP RPRSERASPS ERASPVALTH RALAPHEARG 5040

LEGLGLLEAS NGLNARGLEG LGLNTYRARG METGLNLESE RGLYLELESE RGLNASNGLY 5100

GLNGLYSERL YSLEPHESER ILEPRALAAS PALAGLYASP ASPTYRLYSP RLYSLEPHES 5160

ERTYRLEASP THRGLNLEAS NARGLEPHET YRASNSERLE THRPRALAGL GLNGLNPHEV 5220

ALVALASPAL AILEARGLEL YSASPLEVAL LESERLELEA SNLESERSER PHEASPALAS 5280

ERGLYTYRIL EASPARGLEA SNTHRGLYAL AVALILEPRV ALLEVALARG LEPRASPILE 5340

CYSASNTHRC YSPHELYSLE SERGLNLEGL SERGLYLYSL ESERSERILE ALALEPRARG 5400

LETHRASPLE GLILEASNAR GLETHRGLYA SNLEGLYGLY GLASPTYRGL NASPLYSMET 5460

PRMETPRILE LEVALALAAS PGLYARGASN ALAGLYILEG LNTHRSERAR GASNALAHIS 5520

GLYGLNGLIL ELELEARGAS NALALEGLNT HRMETTYRAS PTHRGLNASP LYSASNASPP 5580

RVALALAVAL PHEASPGLYS ERVALILEPR LYSASNPHEA SPASNASPGL NHISARGASN 5640

PHEGLNGLLE PHEGLYILEL YSASNGLYAS PGLNSERPRP RSERALALEG LYPRLEPRSE 5700

RVALILEGLA RGASNHISAS NVALLESERA LAILEPRGLN GLPRTYRARG ASNHISTHRA 5760

LAGLYILEGL ALAARGASNL EGLTHRGLAR GGLNLEARGA SNLEGLVALL ESERLETHRL 5820

YSASNLEVAL GLYTHRSERG LYPHETHRSE RALAARGASN ASNVALILEI LEGLNLEASN 5880

ARGASNPRAS PLESERSERT HRSERASPTH RTHRASPVAL ILEARGASNS ERILELEGLG 5940

LYPRASPVAL LYSASNSERV ALVALGLYIL ELYSPRTHRV ALGLYLETHR SERARGASNV 6000

ALGLYLEVAL SERVALSERL EASPGLYLYS ASNVALLEAS PTYRGLYALA ARGASNVALV 6060

ALLEASPTHR THRALALESE RALAASNTHR LYSASNTRPH ISARGLESER PHETHRTYRA 6120

SNCYSTHRPR SERALAASNT YRGLNTYRIL EPRALATYRL YSASNTYRPH EALAGLTHRG 6180

LGLNVALMET PHEGLNPRGL YHISILEVAL ARGASNTYRI LEVALVALAS PALAASPSER 6240

SERPRLEGLN ILEVALILEA SPGLYPHEAR GPRPRMETGL ASPILEVALS ERPRTHRLEG 6300

LASPLEILEH ISLEALALEA RGARGPRPRG LNPRASPGLY ALATHRCYSI LELYSGLYAS 6360

NGLCYSGLAL APHECYSVAL ASPGLYVALC YSLYSPRGLN GLYASPPRLY SPRGLNSERI 6420

LEASPTHRIL EVALGLYTHR ASNLEHISME TASPILELES ERASPLEALA ALAALALEAL 6480

AGLYSERILE GLYVALALAP RSERSERASN LEASPPRTHR ARGLYSPRVA LALAASPALA 6540

ALAVALVALA SNALACYSGL SERPHEPRLE SERPHEASPT HRASPVALSE RARGARGPRT 6600

RPVALGLYGL YGLNILEVAL ASNSERILEP RALASERVAL GLLYSILEAL AVALLEGLGL 6660

NVALARGGLN ASPGLYHISP HESERVALPR SERTYRALAG LYHISVALAL ATHRMETTHR 6720

SERVALSERL EARGARGGLN ASPLEPHEGL ALAILEGLAL AGLYARGGLN ILEGLNTHRS 6780

ERARGGLNLE PRPRSERLEP RTYRTHRPHE TYRTHRSERT YRTHRSERGL ASPSERTYRL 6840

YSLYSGLNLE SERGLASPGL YVALASPVAL VALVALVALA LAGLARGARG ASPGLGLHIS 6900

GLNLEASPAS PASPPHEGLG LYILEASPAS PGLMETASPG LSERGLNSER ARGARGPRAS 6960

PLESERTHRP HEPHEALATH RLESERGLIL ESERPRASPG LALAARGARG PRPHEILEIL 7020

EALAARGARG PRGLYASPAR GSERHISSER ARGSERALAA LAALALESER THRSERGLLY 7080

SSERALAALA ALALESERTH RSERGLLYSA SPTRPLEGLN VALARGSERA LAALAASPGL 7140

YLEALASERA LAILETHRSE RLYSSERALA GLYTYRTHRP RLELYSSERA LAMETTHRLE 7200

PRARGSERAL ATHRVALMET THRTYRTRPL YSTHRGLGLG LYALAPRLEP RGLYCYSPRA 7260

RGTHRTHRLE GLNARGSERA SPASPALAIL ETHRARGTHR ASNPRASPAL ALELYSALAS 7320

ERALAVALPR GLYALASERA LALYSSERGL YASNTYRSER TYRGLYVALA RGSERGLYSE 7380

RSERPHESER SERILEGLYS ERALAILESE RMETSERLYS SERGLYSERV ALALALEGLY 7440

LYSSERHISL ESERVALVAL ASPGLYGLYG LASPGLYGLN ASNILEPRLE HISPRLEILE 7500

GLNPRGLARG SERILEALAV ALLYSALAPR SERPRTHRGL PRGLYSERPR ALASERPRGL 7560

YGLYSERGLN PRARGSERIL EILESERARG METLEVALTH RASPPRLYSS ERLEGLYASP 7620

VALCYSMETA SPLEGLNTHR ILETHRTHRS ERSERASPPR ASPPRLYSAR GSERASNILE 7680

THRGLILELE PRALAGLYTH RPRLEPRGLY THRALAALAT HRALAARGGL NASNPRASNP 7740

RALAALAALA ALASERTHRG LYGLYGLNAS PGLYGLYPRA SNASPALAVA LPRARGSERP 7800

RALAALAVAL GLNGLYILET YRGLYASNAR GSERSERGLL YSPRSERILE THRILEASPG 7860

LYASNASNIL EASNLYSSER SERGLYALAV ALTHRGLYGL NSERTHRARG SERSERGLYT 7920

HRGLYTHRSE RTHRGLYALA ALAALATHRG LYTHRGLTHR ASNALAALAS ERVALALALY 7980

SLEGLNMETG LYVALSERAL AALAGLYILE ALAGLYLEAL ALEGLYILET RPALALESER 8040

SERHISPRIL EGLVALPRVA LLYSSERTHR ASPTHRSERI LEASNVALAR GSERVALTHR 8100

SERGLYPHEV ALASPGLYIL ELYSSERVAL THRSERGLYP HEVALASPGL YILELYSASP 8160

GLYLEARGTH RALALEALAA SPTYRALALE CYSALAGLAL ATHRASNMET CYSARGTHRA 8220

LASERASNPH EASPGLNPRH ISSERASPGL SERALALEGL NHISLEARGT HRALAVALPR 8280

ILEASNGLYP RASPSERPRG LYTHRPRGLG LYVALLYSTH RASPTYRSER VALCYSGLYG 8340

LTHRTHRILE PHELYSTHRG LYTYRVALAS NTYRASNVAL ASPTHRTHRA SNLEARGTHR 8400

ILEPHEGLYT RPASPILEAL AGLGLYGLNL YSTHRILESE RASNVALVAL ASPASNGLLE 8460

ALAARGTHRI LEVALSERPR ASPGLYPHEA SNTRPASPTY RGLYSERTHR ARGTHRLEGL 8520

YILEASPILE ALAARGTHRL ESERTHRASN GLGLGLYTYR GLTHRSERAL AVALARGTHR 8580

METLEVALGL YMETASPVAL THRHISPRSE RPRGLYSERS ERALAASNAL APRSERVALA 8640

LAGLYMETVA LALASERVAL ASPSERTHRL ESERGLNTRP PRALAGLILE ARGVALGLNA 8700

RGTHRASNTH RGLNVALPRA SPALACYSTH RGLNCYSPHE GLNLYSTHRG LNGLYPRHIS 8760

SERTHRPHEA SPARGTHRSE RGLYSERGLY SERSERSERP RGLPRARGTH RTHRASPVAL 8820

GLYTHRPHEG LYGLNLYSTH RTHRGLYALA PHEASPGLSE RGLYPRPRLE SERGLNLYST 8880

HRTHRASNGL YILEVALSER THRASNGLSE RGLYARGTHR THRSERGLNT RPASNVALLE 8940

ASPLELYSTH RTHRTHRLEA SPGLNGLYHI STYRGLNSER ARGTHRTHRT YRASNVALVA 9000

LALAGLNTHR LYSTHRVALA SNVALASNAS NLELYSVALA LALEVALTYR GLYASPARGV 9060

ALALATHRIL EGLYSERALA THRPHEALAA RGVALCYSAS NLEILEGLYL EMETGLYLEA 9120

RGVALASPPH ETYRASNASN LELYSVALGL LEGLNSERLY SVALGLARGT HRGLYTYRAL 9180

AALAPHEARG VALGLSERAL ASERALAASP LEILESERTH RILETHRLYS VALPHEASPA 9240

LAGLYHISTH RVALPRALAP HEGLNPRGLT HRMETPHEAR GVALPHEGLA LAGLYHISGL 9300

VALPRALATY RGLNPRGLTH RALATYRGLI LEPHEHISAR GVALGLYGLG LGLTHRPRAL 9360

ALEVALHISA SPLEASNTHR ALAMETARGV ALGLYPHELE ALASERVALG LTHRPRALAS 9420

ERILEGLALA ALASERGLLE SERLYSVALG LYGLYTHRLE ALATYRVALS ERVALGLILE 9480

GLLYSVALLY SVALGLYTHR ILEILETHRG LYASPPRLEA SPPRPRVALL ELYSVALILE 9540

PRLEGLNGLY CYSASPALAA SPGLTYRGLY ARGVALLELE HISPRLELET HRALAALAAL 9600

ALELEGLYAL ASERALAARG ALAGLNSERV ALVALGLYTH RPRPHEGLYP HEALASERGL 9660

YTHRTHRGLY GLYGLYASNA LAALAPRALA ALAPRLYSVA LASNGLYVAL GLTYRGLYGL 9720

THRARGVALG LNLEASPGLG LYLELYSARG VALSERILET RPTHRGLSER TYRGLYGLYA 9780

RGVALSERAS NASPLEALAA RGVALSERGL NILESERGLY ASNARGPRLE ASPALALEAS 9840

PGLNGLYTHR ARGVALSERT YRTHRGLTYR ASPSERTYRT YRASPHISTY RASNLYSVAL 9900

THRASNSERP RSERASNLEV ALTRPTYRSE RILESERTHR ARGVALVALA LAVALASPTH 9960

RALASERASN LYSVALVALA SNTYRTYRSE RASPASPPRT HRGLYMETSE RASPSERGLY 10020

GLASPALAPH EASPMETARG LYSVALTYRA LATHRPRASP GLNASPILEG LHISGLYARG 10080

TRPASNGLTH RILETYRVAL ILEILETHRS ERPHESERAS PTHRLETHRI LEGLNPRTYR 10140

ASPTRPASNG LPHEARGLYS TRPASNPHEI LEMETASNSE RARGTRPARG HISTYRTYRL 10200

EARGTYRALA GLYGLTYRGL PHEGLNALAA SPLEPHELYS TYRCYSALAS ERALAGLNGL 10260

ASPASNALAT HRLEGLNALA LELEARGTYR ASPLEASNLE GLASNLYSTY RGLYPRSERP 10320

HETHRALAPH EPHEGLNGLG LNASNGLLYS TYRLYSGLPR GLYALAGLGL YVALCYSGLT 10380

HRTHRPRGLY VALLYSTYRL EALASERTHR GLNMETGLPR THRASPALAA RGTYRLEASP 10440

GLNGLNILET HRALAGLTHR LYSTYRLEAS PTHRLEPRGL ILELYSTYRL ETHRASNSER 10500

GLNALALEAL AASPLEPRTY RPHEALAGLL YSTYRLEVAL ASPGLNLEAS NPRGLGLYLY 10560

STYRGLNGLY ALASERGLNC YSPRPHEARG TYRGLNPRHI STHRVALTHR THRVALSERA 10620

LAGLYALASE RASPPRARGG LYSERPRGLG LYGLYGLYAR GTYRVALASP ALAGLYGLYP 10680

HEGLPRSERI LELYSTYRVA LTHRSERASN ALAVALSERV ALGLYVALTH RHISPHEALA 10740

GLYSERARGA LAALAALALE ALAGLLEVAL TRPSERGLYA SNARGALAAL AALAPRLYSS 10800

ERALAALALE ASPALALEGL NGLNSERILE TYRLEGLNPR LYSALAALAT HRTYRCYSPR 10860

GLASNILEGL LYSALAGLAS PTYRLELEAS NPRSERPRLY SALAGLHISC YSPHEASPTY 10920

RASPLESERT YRLYSPRALA ASPLYSALAG LASNGLNALA VALALAVALG LYARGALAGL 10980

YALAVALALA ALAVALVALT YRASNASNGL LYSALAGLYL YSPRTHRLEG LYPHELEASN 11040

PRLELETYRS ERGLYALALE LYSALAGLYS ERSERPRTHR ASPILEILES ERGLYILESE 11100

RASPLYSALA ILEHISASPG LVALSERPRV ALGLYASPTH RASPALALEL EGLARGALAI 11160

LEMETGLYAL AGLGLALAAL ALYSALALES ERGLMETILE LEGLNSERGL LYSALALEVA 11220

LGLGLYSERT HRPHEALALY SALALETYRS ERSERALAAL ATHRGLYTHR TYRALASERS 11280

ERTHRTHRVA LTYRLYSALA ASNGLGLNPR THRTRPVALT YRARGALAAS NPHEGLVALG 11340

LTHRPRARGA LAASNASNTY RCYSSERASN GLNVALGLGL YPRTYRSERL ETYRSERGLY 11400

ARGALAPRVA LVALGLNTYR ALALEASNAR GALAGLNASN ASPPRASNAL APHEGLYVAL 11460

VALALAALAA RGALASERAL AILEGLNLEA SPGLYILEIL ETYRARGALA SERMETVALT 11520

RPGLGLALAG LNGLNVALSE RGLYLYSALA SERASNSERL EGLNTYRVAL ASNVALGLNV 11580

ALLYSALATH RGLYASPVAL LEPHEASNTH RLYSALAVAL GLYGLNALAT HRGLARGALA 11640

VALHISGLAS PLEASPVALA LAALAILEAS PALAALAGLV ALARGALAVA LLELELEASP 11700

GLALAASPVA LPHEMETGLG LARGALAVAL SERPRSERPH EGLASPVALT RPSERGLNPR 11760

ARGCYSGLNS ERVALPHEAS NPRASNILEP RLYSASPALA TYRSERPRHI SGLILETYRS 11820

ERARGASPPH ETHRASPILE THRALAGLYS ERSERILEGL YCYSASPGLY VALASNPRGL 11880

NTHRGLYLYS ASPGLYLEGL GLYSERPHEL YSASPLYSAS PPRGLLYSAS PPRLYSALAI 11940

LEGLLEPRAR GASPGLNILE ILEGLCYSAR GASPSERGLY LEVALMETLY SASPSERPRL 12000

ETYRPRTYRA RGASPVALHI SGLYPHEALA THRARGASPV ALLYSSERME TLYSASPVAL 12060

VALVALVALG LYGLYGLYAL ASERGLYALA TYRALAALAV ALARGASPTY RGLNVALGLM 12120

ETVALASNLY SGLALAGLYL EVALPRPHEG LNVALSERPR THRTHRLYSG LGLPRSERPH 12180

EGLNPRASPA SPVALTHRLE LELESERGLN ASPPRGLYHI STRPGLLYSG LGLYILESER 12240

ILEHISTHRC YSASPGLNAR GGLHISHISG LLEALAILEA LASERLYSGL ILEPRVALGL 12300

YTYRSERALA ALAASPILEA SPTHRASNAR GGLLEASPTH RGLNHISILE HISPRPRASP 12360

SERTYRPHEV ALSERPRLET HRARGGLPRG LYILECYSGL THRTHRPRGL YVALLYSGLP 12420

RSERASNASP PRASNPRPRG LTHRTYRSER LYSGLSERLE GLASPILEAR GLYSGLTYRL 12480

EVALALAASN GLYVALGLNA LAGLNALALE VALPRLYSPH EGLPRPRALA VALTYRASNA 12540

SPGLLELYSP HEGLYALATH RGLYASPGLT YRARGPHEGL YLYSPRVALG LYALAVALGL 12600

YSERALAALA THRALALELY SPHELEASPG LALALETHRT YRPRPRPRLY SPHEASNVAL 12660

ASPGLTHRAL APHETHRGLY ALATRPGLYA RGPHEARGGL NASPLEILES ERGLILELYS 12720

PRCYSCYSGL GLLYSPHETH RALAVALPHE THRPRSERIL EVALGLARGP HETHRASPTH 12780

RPRVALLETY RGLYPRLYSP HEVALTHRAS PASNGLYASP SERLYSPHEV ALTHRASNME 12840

TGLNALAALA LELELYSGLY PHEPRASPVA LALAALAHIS SERLETHRPR ARGGLYGLYS 12900

ERILELEPRM ETGLNGLVAL ALALETHRTH RARGGLYILE ASPVALALAL YSPRTHRGLY 12960

ARGGLYILEM ETLEASPTHR GLYARGGLYL YSGLSERCYS LYSGLYLETY RALAGLYHIS 13020

ARGGLYMETV ALPHESERIL EASPALAGLN GLYGLLYSGL YPRALAARGA RGARGGLYGL 13080

NLEGLYPHET RPGLYASNLY SGLYSERILE VALGLYPRAR GTRPLYSLEP RPHEMETGLY 13140

PRPHELEGLN SERVALASNP RLYSGLYTHR VALPHEPRSE RGLTHRGLGL YGLSERMETA 13200

LASERARGGL YVALASPPHE THRGLASPPR LELEGLNGLY ARGGLYVALL YSILESERGL 13260

YTRPASPVAL GLTHRLEGLY ASPGLILETH RHISVALGLY GLLYSPHETH RLYSGLYTYR 13320

LYSPRSERAL ASERSERGLY SERLYSHISA LAGLYGLNCY SGLYGLTYRH ISGLASNLYS 13380

HISPHETHRS ERLEGLGLLY SHISPHEVAL ASPTHRPHEG LYLEHISGLY HISLYSHISG 13440

LYGLYPRASN PHEGLGLNLE PRILEASNGL NPRARGHISG LYILEPRGLY GLYGLYILEA 13500

LATHRGLYAL AGLGLYILEL YSHISMETPH EGLYLEVALA LASERGLASP ALAGLYARGH 13560

ISPRVALGLV ALALAGLGLG LALASERLYS PRLYSHISVA LASPGLYPHE GLYILEHIST 13620

HRPHEARGHI SVALGLNLEL EGLNLEASNM ETGLTYRASP ASPASPILEL ECYSARGLYS 13680

SERLYSHISV ALTYRASPAL AVALGLNASP LYSILEALAP HEALASERTY RLEGLGLTYR 13740

ALAARGILEA SPALATHRTH RASNPRGLYM ETARGILEAS PTYRILEGLY GLYGLYASPL 13800

EPHEARGILE GLILEGLASN SERILEARGI LEGLASNGLN SERASPALAA SPGLYTYRSE 13860

RSERCYSSER THRLELYSIL EPHEGLGLNL EGLGLYMETS ERLESERLYS ILEPHESERT 13920

YRLYSMETAS NSERTHRLEA RGTYRLEPRP HEARGILEGL YLEHISPHEA RGTHRARGIL 13980

EHISLETHRV ALPRGLASPL EARGILEGLN ASPGLYSERG LNVALLYSIL EGLNGLYILE 14040

SERASNPRSE RGLYALALES ERSERGLYGL YLEGLYGLPR LYSILEGLNS ERLYSLEARG 14100

GLYLEVALGL NARGILEARG ASPALAMETA RGGLNARGIL EARGLEHISL EGLARGTHRG 14160

LYGLNLEGLY VALGLYSERA SPGLYASNPR VALVALALAG LYARGILESE RALATYRVAL 14220

GLGLYSERSE RARGILEVAL PRGLGLTYRV ALPRILETHR LYSILETYRS ERPHEPHEVA 14280

LGLYGLYALA VALPRGLASN LEARGILETY RVALTHRGLY GLSERTYRAL AGLYARGLYS 14340

ASPILEARGH ISGLYHISLY SLYSGLHISA SPLYSSERLY SPHETHRASP THRPRVALLE 14400

TYRGLYPRLY SLYSGLYASP ALAPRTHRIL EASPTHRSER ASNTYRPHEL EPHEGLYLYS 14460

LYSMETGLAS PASPLEARGL YSTYRTHRVA LPRSERTHRC YSGLYVALLY SLEGLMETTY 14520

RGLNGLYGLY ILEGLLESER ALALELEGLN METILEGLNA SPALAILEAR GLEPHESERT 14580

YRLEASPTHR GLNLEASNAR GLEGLYILET HRTYRTHRTH RTYRSERLYS LELYSASPLE 14640

VALLESERLE LEASNALALE GLNGLYGLYA RGLEASNTHR GLYALAVALI LEPRVALLEV 14700

ALARGLEASN VALILEASPP HEPRLYSLEG LNVALARGAL AALAALAARG ARGLESERAL 14760

AGLYSERARG LESERGLLEG LTRPILEARG LESERGLNLE GLSERGLYLY SLESERSERI 14820

LEALALEPRA RGLETHRASP LEGLILEASN ARGLEVALAL AHISSERVAL ALATHRTYRA 14880

LAARGLEVAL CYSPHEPHEP RTHRLYSLEV ALGLNASNAS PPHEASNTHR LELEARGMET 14940

ALAPRMETSE RGLGLASPLE ALATRPPHEA RGSERTHRPH EHISPRILEP RLYSMETGLY 15000

SERLESERAS PVALARGMET LYSSERILEG LGLLYSGLYG LGLYMETTHR ASNASPTYRI 15060

LESERALALE THRLYSASNA LAPHEILETH RASNTYRPRS ERGLGLNARG ASNALAGLYI 15120

LEGLNTHRSE RARGASNPHE SERARGPRLY SASNHISGLY THRSERTHRV ALALAPRGLN 15180

VALGLNALAS ERVALTYRAR GASNHISASN VALLESERAL AILEPRGLNG LPRTYRARGA 15240

SNILEASNME TLELETYRGL YTHRASPASP CYSSERGLYL YSASNLEASP GLLETRPILE 15300

VALGLYHISG LYALAVALAL AARGASNLEV ALGLYTHRSE RGLYPHETHR SERALAARGA 15360

SNMETHISAS PVALILEGLY ASNASPGLYT HRVALPRSER GLPHEARGAS NASNVALILE 15420

ILEGLNLEAS NARGASNSER METTHRASPC YSCYSILEGL THRTYRLEME TLYSSERGLA 15480

RGASNTHRLE ALAPHEPHES ERGLYASNGL VALILEASNA SPGLYPRSER SERLYSASNT 15540

HRPRVALPRS ERCYSPHEHI SPHEPHEILE TYRLYSGLYC YSTRPMETPH ELETYRARGA 15600

SNTYRPHEAL AGLTHRGLGL NVALMETPHE GLNPRGLYHI SILEVALARG PRASPGLYTH 15660

RGLYPHEARG LYSPRLETRP ARGHISTYRP HEGLNASNTH RGLNGLYILE ILEPHEVALV 15720

ALASPSERAS NASPARGPRV ALVALGLNVA LLEMETPRGL GLYMETASPS ERASPGLSER 15780

GLNALAILEL EASNASNILE GLYALAASPG LYGLNSERAL AGLNGLYALA SERPRGLYVA 15840

LVALILEALA SERPRSERLY SGLNASPLEP HEGLALAILE GLALAGLYAR GGLNTHRTYR 15900

ALASERCYST RPGLYGLYVA LGLYGLNGLY GLCYSARGGL YSERSERASN CYSLYSARGC 15960

YSTRPSERGL YVALPHETYR SERASNTRPI LEGLNGLLEL EARGARGSER METGLYLEGL 16020

SERARGSERA LAALAASPGL YLEALASERA LAILETHRSE RLYSSERALA ILESERGLNT 16080

YRGLYASPSE RPHEALALYS SERALAMETT HRLEPRARGS ERALAVALGL NSERASPVAL 16140

TRPARGSERA SPGLYGLNCY SSERASPLEL ELYSSERASP LYSLEASNVA LILEASPPHE 16200

PRLYSSERAS PTYRASPALA PHEILEARGS ERASPTYRGL NGLCYSALAA SPALAPRGLY 16260

GLNLYSSERA SPTYRSERAL ALEGLNSERG LNGLYLEILE LESERLEARG SERGLMETLE 16320

ALAGLGLNAS PLYSSERGLS ERASNPRGLY VALMETSERT HRARGSERGL YALAASPTHR 16380

HISLYSSERI LEVALILEAR GSERILETYR ALAILEASNS ERGLYARGSE RLEPRLEILE 16440

VALGLYASNS ERASPGLNGL GLYLYSSERG LNSERASPPH EGLSERGLPH ESERTHRALA 16500

LYSSERSERG LYALAVALTH RGLYGLNSER THRARGSERS ERSERALATY RGLSERLETH 16560

RSERALAVAL LYSSERTHRA SPTHRSERIL EASNVALARG SERVALVALG LASNASNASN 16620

ASPGLYLETH RALAALATYR ARGTHRALAL EPHEASPSER HISGLTYRAR GTHRALASER 16680

ASNPHEASPG LNPRHISSER ASPGLSERAL ALEGLNHISL EARGTHRCYS HISARGCYSC 16740

YSTHRTHRPH EALAPRASPA LATHRGLCYS GLASNCYSLY SHISTHRARG THRASPTYRS 16800

ERVALCYSGL YGLTHRTHRI LEPHELYSTH RGLYGLTHRT HRGLNILEHI SALAARGTHR 16860

GLYPRSERIL EGLNASPARG THRILESERA SNVALVALAS PASNGLLEAL AARGTHRLYS 16920

SERLEPRARG THRLEPRPRL EGLNTYRARG ASPLEASPLE LEPRLEHISG LNASNLEILE 16980

LYSTHRLEVA LSERTHRGLY ARGTHRPRAL AALAHISARG ALAARGTHRS ERGLYSERGL 17040

YSERSERSER PRGLPRARGT HRTHRASPVA LGLYTHRPHE GLYGLNLYST HRTHRGLMET 17100

THRGLNARGT HRTHRSERAS NPRGLTHRAR GTHRVALGLY SERSERCYSP RTYRCYSASP 17160

SERGLNALAP RGLNVALARG THRVALASNG LYGLYPHEGL NILEALAARG THRVALASNV 17220

ALASNASNLE LYSTHRVALT YRALAPHEAS PVALSERGLA SPGLYSERTY RLELYSTHRT 17280

YRGLVALVAL GLYASNVALT YRLYSVALAL ALEVALTYRG LYASPARGVA LALAPRASNS 17340

ERGLYALATY RLEASNGLAL AASPPHEARG VALALASERL ELEGLNARGV ALALATHRIL 17400

EGLYSERALA THRPHEALAA RGVALCYSAS NLEILEGLYL EMETGLYLEA RGVALASPAS 17460

NVALVALALA SERPHELYSV ALGLTYRSER ASPALAALAL YSVALPHEGL ALAGLYHISG 17520

LVALPRALAT YRGLNPRGLT HRALATYRGL ILEPHEHISA RGVALGLYSE RILEGLPHET 17580

HRALALEPRG LNLEGLNSER LEASPPHETH RLYSVALILE PRGLILEASP METPRSERHI 17640

SSERSERSER GLYTRPLYSV ALLEASPARG ASPPRASNHI SALALYSVAL LEPHELEGLY 17700

ARGVALLEIL EALAASPMET CYSARGARGV ALLEPRGLNV ALILEGLALA THRASNARGV 17760

ALGLNASNGL YALAVALTHR TRPGLSERAS PPRASNARGV ALGLNASNGL YALAVALTHR 17820

TRPGLSERAS PPRASNARGL YSVALSERAS NASPLEALAA RGVALTHRAL AMETARGTYR 17880

TRPTRPLECY SGLILEALAT YRCYSPHEAL ASERVALGLY GLYLYSVALV ALTHRASPSE 17940

RPHEARGVAL TYRSERVALA SPASNSERLY STRPASPASN LEASPSERAL AALALEASNT 18000

HRLYSTRPPH EALAGLASPP RSERARGTYR CYSALASERA LAGLNGLASP ASNALATHRL 18060

EGLNALALEL EARGTYRCYS GLYVALGLYV ALASNILELE TYRGLARGTY RGLALAALAI 18120

LEGLNGLYVA LALAALATHR ASPLYSTYRP HETYRGLYAS PASNTYRALA THRLEARGTY 18180

RGLYALATYR SERVALCYSS ERPRLYSTYR GLYGLTHRGL LYSSERGLYL EGLSERILEA 18240

LAALAALAAR GTYRILEALA ARGPRASPIL EMETLYSTYR LEASPGLNGL NILETHRALA 18300

GLTHRLYSTY RLEVALASPG LNLEASNPRG LGLYLYSTYR GLNPHEPRGL NTHRPRSERA 18360

RGTYRARGHI SLEPRPRGLT HRVALTHRGL YILELEGLYA RGALATHRPH ETRPTRPILE 18420

ASNSERILEL ELYSTYRTHR ALAGLGLYTY RGLALAALAT HRLYSTYRVA LASPALAGLY 18480

GLYPHEGLPR SERILELYS 18499

Claims (10)

1. A method for rapidly identifying the relative abundance of aflatoxin-producing bacteria in farmland is characterized in that: the method comprises the following steps:

(1) Culturing a soil sample to be detected by using a Chlamydia medium or other culture mediums suitable for the growth of the toxic aspergillus flavus, and preparing a liquid to be detected of the soil sample to be identified for later use;

(2) The method for identifying and comparing the relative abundance of the aflatoxin-producing bacteria in farmland soil by adopting an indirect non-competitive double antibody sandwich method comprises the following steps:

a, adding a liquid to be tested into a hole of an enzyme-labeled plate coated with a nanometer antibody or a monoclonal antibody of a molecule AFT-YJFZP008 for indicating the toxicity of aflatoxin toxigenic bacteria, reacting, and washing the plate;

b, adding a molecular AFT-YJFZP008 polyclonal antibody for indicating the toxicity of aflatoxin toxigenic bacteria for reaction, and washing the plate;

c, adding a horseradish peroxidase labeled antibody which is combined with a molecular AFT-YJFZP008 polyclonal antibody for indicating the toxicity of aflatoxin-producing bacteria, reacting, and washing the plate;

d, adding a color development liquid for reaction; adding a stop solution, and reading and calculating a result by an enzyme-labeling instrument;

(3) By comparing the AFT-YJFZP008 concentration, the relative abundance of the aflatoxin-producing bacteria in the farmland soil is known, namely the abundance order, namely the determined AFT-YJFZP008 concentration and the relative abundance of the aflatoxin-producing bacteria in the soil are in positive correlation, namely the higher the determined AFT-YJFZP008 concentration is, the higher the relative abundance of the aflatoxin-producing bacteria in the soil is, and the higher the risk of the corresponding farmland-producing bacteria of the soil sample is;

the molecule indicating the virulence of aflatoxin-producing bacteria refers to AFT-YJFZP008 peptide, and the amino acid sequence of the molecule is shown as SEQ ID NO. 1.

2. The method according to claim 1, characterized in that: and replacing the solution to be tested with a serial concentration of molecular AFT-YJFZP008 pure product solution which plays a role of a standard substance and indicates the toxicity of aflatoxin toxigenic bacteria, and preparing a standard curve, and calculating to obtain the concentration of AFT-YJFZP008 in the sample to be tested.

3. The method according to claim 1, characterized in that: the method comprises the following steps of: weighing a soil sample to be detected, transferring the soil sample to a sample diluent, vibrating at room temperature until the soil sample is uniform, preparing a uniform dispersion of the sample to be detected, taking 10-1000 mu L of the uniform dispersion of the sample to be detected, adding the uniform dispersion of the sample to be detected into a conventional liquid culture medium containing 6-600mL, placing the culture medium into a 200+/-50 rpm vibration culture medium at 15-35 ℃, and sampling after culturing 6-24h to form the sample to be detected of the sample to be identified.

4. A method according to claim 3, characterized in that: the sample diluent is 0.01mol/L phosphate buffer solution containing 0.1% sorbitol and 0.1% soft sugar.

5. The method according to claim 1, characterized in that: the hole bottom is coated with a nano antibody or a monoclonal antibody of a molecule AFT-YJFZP008 for indicating the toxicity of aflatoxin toxigenic bacteria, and the preparation method comprises the following steps: dissolving the AFT-YJFZP008 nano antibody or monoclonal antibody in ELISA coating buffer solution to form 0.2-8.0 mug/mL coating solution, adding the coating solution into an ELISA plate, standing overnight at 4 ℃ or standing at 37 ℃ for not less than 2h, removing the coating solution in the ELISA plate, and washing the ELISA plate with ELISA conventional washing liquid; then adding ELISA routine blocking solution, standing at room temperature or 37 ℃ to block at least 1h, discarding the blocking solution, and washing the ELISA routine washing solution.

6. The method according to claim 1, characterized in that: the specific steps of the step (2) are as follows:

a, adding 100-200 mu L of the liquid to be tested into a hole of an enzyme-labeled plate coated with a nanometer antibody or a monoclonal antibody of a molecule AFT-YJFZP008 for indicating the toxicity of aflatoxin-producing bacteria at the bottom of the hole, standing at room temperature or 37 ℃ to react at least 1h, discarding the reacted liquid, and washing the enzyme-labeled plate;

b, adding AFT-YJFZP008 rabbit-source polyclonal antibody, standing at room temperature or 37 ℃ to react at least 1h, discarding liquid, and washing the ELISA plate;

c, adding horseradish peroxidase labeled goat anti-rabbit antibody, standing at room temperature or 37 ℃ for reaction of not less than 1h, discarding liquid, and washing the ELISA plate;

and d, sequentially adding ELISA color development liquid and stop solution, and finally reading and calculating the concentration of AFT-YJFZP008 in the sample to be detected by an enzyme-labeling instrument.

7. The method according to claim 6, wherein: the polyclonal antibody of the molecule AFT-YJFZP008 which indicates the virulence of the aflatoxin-producing bacteria is different from the animal source of the nano antibody or the monoclonal antibody of the molecule AFT-YJFZP008 which indicates the virulence of the aflatoxin-producing bacteria.

8. The method according to claim 6, wherein: the ELISA chromogenic liquid refers to conventional hydrogen peroxide and TMB chromogenic liquid for ELISA.

9. The method according to claim 6, wherein: the stop solution refers to a conventional chromogenic stop solution for ELISA: 2mol/L sulfuric acid aqueous solution.

10. Use of the method of any one of claims 1-9 for risk monitoring of the occurrence of toxigenic bacteria of aflatoxin in the field.