CN113820491A - Early risk early warning intelligent sensing card for aflatoxin pollution and application thereof - Google Patents

Early risk early warning intelligent sensing card for aflatoxin pollution and application thereof Download PDF

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CN113820491A
CN113820491A CN202111041457.3A CN202111041457A CN113820491A CN 113820491 A CN113820491 A CN 113820491A CN 202111041457 A CN202111041457 A CN 202111041457A CN 113820491 A CN113820491 A CN 113820491A
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aflatoxin
early
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CN113820491B (en
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李培武
张奇
姜俊
唐晓倩
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Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
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Abstract

The invention discloses an aflatoxin pollution early risk early warning intelligent sensing card and application thereof. The aflatoxin early warning molecule comprises an aflatoxin early warning molecule nano antibody or an aflatoxin early warning molecule monoclonal antibody, an aflatoxin early warning molecule rabbit polyclonal antibody marked by a signal material, a sample pad, a nitrocellulose membrane, a water absorption pad, a bottom plate and a goat anti-rabbit antibody, wherein the aflatoxin early warning molecule is AFT-YJFZP008 peptide, and the amino acid sequence of the aflatoxin early warning molecule is shown in SEQ ID No. 1. The method is used for rapidly sensing the aflatoxin production capacity of aspergillus toxin-producing aspergillus flavus strains, identifying whether aflatoxin toxin-producing strains with strong toxicity exist in farmland, agricultural products, traditional Chinese medicinal materials, feeds and other products, intelligently sensing the aflatoxin pollution risk level in the agricultural products, and is easy to popularize and apply.

Description

Early risk early warning intelligent sensing card for aflatoxin pollution and application thereof
Technical Field
The invention relates to an aflatoxin pollution early risk early warning intelligent sensing card and application thereof.
Background
The aflatoxin has strong toxicity and great harm, is a pollutant which pollutes most food types, is one of the most serious countries in the world with aflatoxin pollution, generally presents a pollution aggravating trend in recent years, and seriously threatens food safety and people health. Aflatoxin is a kind of mycotoxin with the highest toxicity in nature, wherein aflatoxin B1 is a class I carcinogen identified by International Agency for Research on Cancer (IARC), has caused many cases of poisoning of human and livestock, and is one of the main causes of high incidence of liver Cancer cases. According to the statistics of the Web of Science retrieval data in the last 5 years: the aflatoxin-polluted food and raw material variety exceeds 110, and the aflatoxin-polluted food and raw material is high in the first place of pollutants.
Aflatoxins are produced mainly by toxigenic fungi such as aspergillus flavus. Taking peanuts as an example, the peanuts are already provided with toxigenic fungus strains of aflatoxin in the field, the strains enter a packaging bag, a transport vehicle, a warehouse, a processing line and the like together with the peanuts after being harvested, and once the conditions are proper, a large amount of aflatoxin can be generated, so that the consumption safety of the peanuts and products thereof and the life health safety of people are threatened. The agricultural research bureau of the department of agriculture of the United states, the International research institute for semiarid of the Food and Agriculture Organization (FAO) of the United nations, and other international well-known research institutions, classify the virus-producing fungi of aflatoxin as soil-borne pathogens of crops such as peanuts and the like for research, prevention and control.
The existing research shows that the difference of the virulence production of the aflatoxin-producing fungi among different strains is large, and the strains from the non-producing fungi to the high-producing virulence strains are distributed. Taking Aspergillus flavus strains separated from peanuts as an example, a certain part of the Aspergillus flavus strains lack a toxin production gene or a key toxin production regulation gene, and the strains do not have the capability of producing aflatoxin; less than about 20% of the Aspergillus strains are virulent strains. How to assess early warning risk in advance of aflatoxin contamination? This has been a worldwide problem in the art.
Aiming at the problems, through systematic research for ten years, an inventor team discovers an indicator molecule for generating toxicity of the toxin-producing fungi of the aflatoxin, and confirms that the indicator molecule has an aflatoxin pollution early warning function for the first time, so that the indicator molecule becomes an aflatoxin early warning molecule, and researches discover that the occurrence level of the early warning molecule and the aflatoxin pollution level have obvious positive correlation, so that by utilizing the correlation, the early risk warning intelligent perception card for the aflatoxin pollution of agricultural products is further invented and used for early risk warning of the aflatoxin of the agricultural products, and scientific basis is provided for source prevention and control and early prevention and control of the aflatoxin of the agricultural products.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an early aflatoxin pollution risk early warning intelligent sensing card, and application thereof, wherein the early aflatoxin pollution risk early warning intelligent sensing card is used for quickly sensing the aflatoxin production capacity of aspergillus toxin-producing aflatoxin strains, identifying whether strong-toxicity-producing aflatoxin virus-producing strains exist in products such as farmlands, agricultural products, traditional Chinese medicinal materials, feeds and the like, intelligently sensing the aflatoxin pollution risk level in the agricultural products, and is easy to popularize and apply.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the aflatoxin pollution early risk early warning intelligent perception card comprises an aflatoxin early warning molecule nano antibody or an aflatoxin early warning molecule monoclonal antibody, an aflatoxin early warning molecule rabbit polyclonal antibody marked by a signal material, a sample pad, a nitrocellulose membrane, a water absorption pad, a bottom plate and a goat anti-rabbit antibody, wherein one surface of the bottom plate is sequentially pasted with a water absorption pad, a detection pad and the sample pad from top to bottom, the adjacent pads are connected in a joint in an overlapping way, the detection pad takes the nitrocellulose membrane as a base pad, a transverse quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with the goat anti-rabbit antibody, the detection line is coated with the aflatoxin early warning molecule nano antibody or the aflatoxin early warning molecule monoclonal antibody, and the aflatoxin early warning molecule rabbit polyclonal antibody marked by the signal material is loaded on the sample pad, or the aflatoxin early warning molecule is AFT-YJFZP008 peptide, and the amino acid sequence of the aflatoxin early warning molecule is shown in SEQ ID NO. 1.
According to the scheme, the signal material refers to conventional europium latex microspheres, gold nanoparticles and other marking materials which can achieve similar effects, and can be obtained commercially.
According to the scheme, the aflatoxin early warning molecule rabbit polyclonal antibody marked by the signal material is a product obtained by coupling the signal material with the aflatoxin early warning molecule rabbit polyclonal antibody by a conventional marking method.
The sample pad, the nitrocellulose membrane, the absorbent pad, the bottom plate and the goat anti-rabbit antibody are conventional materials of an immune test strip and can be obtained by commercial purchase.
The application of the early aflatoxin pollution risk early warning intelligent perception card is provided, and comprises the following steps: the method is used for rapidly sensing the capability of Aspergillus strains for producing aflatoxin, or identifying whether aflatoxin toxin-producing strains with strong toxicity exist in products such as farmlands, agricultural products, traditional Chinese medicinal materials and feeds, or intelligently sensing the aflatoxin pollution risk level in the agricultural products.
The aflatoxin toxin-producing strain for rapidly sensing the aflatoxin-producing capability of the aspergillus strain and identifying whether products such as farmlands, agricultural products, traditional Chinese medicinal materials, feeds and the like have strong toxin-producing capability or not is applied to the field, agricultural products, traditional Chinese medicinal materials, feeds and the like, and the application method comprises the following steps: culturing and preparing a to-be-detected liquid of a strain to be identified or a sample to be identified by using a conventional Czochralski culture medium, and then determining the content of aflatoxin early warning molecules AFT-YJFZP008 by using the aflatoxin pollution early risk early warning intelligent perception card, wherein the specific method comprises the following steps: when the aflatoxin early warning molecule rabbit polyclonal antibody marked by the signal material is loaded on the sample pad, adding a strain to be identified or a liquid to be detected of a sample to be identified onto the sample pad of the aflatoxin pollution early risk early warning intelligent perception card, reacting for a period of time, and reading an analysis result;
the method comprises the steps of loading no aflatoxin early warning molecule rabbit polyclonal antibody marked by a signal material on a sample pad, adding a strain to be identified or a liquid to be detected of a sample to be identified into the aflatoxin early warning molecule rabbit polyclonal antibody marked by the signal material, then inserting an aflatoxin pollution early risk early warning intelligent sensing card into the strain to be identified or the liquid to be detected of the sample to be identified, reacting for a period of time, and reading an analysis result.
According to the scheme, the application method further comprises the following steps: and (3) evaluation of a detection result: according to the measurement result of the aflatoxin pollution early risk early warning intelligent perception card, the content of aflatoxin warning molecule AFT-YJFZP008 in the liquid to be measured of the strain per unit volume is obtained, and the aflatoxin production toxicity of the strain to be identified is analyzed. The higher the concentration of aflatoxin early warning molecule AFT-YJFZP008 in the strain to be identified is, the stronger the aflatoxin production capability, i.e. the production toxicity, of the identification strain is;
according to the measurement result of the aflatoxin pollution early-stage risk early-stage warning intelligent perception card, the content of aflatoxin warning molecule AFT-YJFZP008 in unit volume of sample liquid to be identified is obtained, and whether the sample to be identified contains a strong-virulence-producing strain of aflatoxin is judged. And (3) if the concentration of aflatoxin early warning molecule AFT-YJFZP008 in the sample to be identified is high, indicating that the sample to be identified contains a strong-virulence-producing strain of aflatoxin.
According to the measurement result of the aflatoxin pollution early-stage risk early-stage warning intelligent perception card, the content of aflatoxin warning molecules AFT-YJFZP008 in a unit volume of sample liquid to be identified is obtained, and the aflatoxin pollution risk in agricultural products is predicted.
According to the scheme, the preparation of a solution to be detected of the strain to be identified comprises the following steps: culturing and diluting the strain to be identified to obtain a solution to be detected of the strain to be identified; the method specifically comprises the following steps: culturing the strain to be identified in a conventional Chaudou culture medium or other culture media suitable for the growth of the strain, wherein the culture environment temperature is 15-35 ℃, and the culture time is not less than 12 hours, then fully homogenizing the mixture of the culture medium and the culture, and diluting by 1-10 times with sterile water to obtain a solution to be detected of the strain to be identified;
preparing the sample solution to be identified: culturing a sample to be identified to obtain a liquid to be detected of the sample to be identified; the method specifically comprises the following steps: weighing a sample to be identified, transferring the sample to sterile water, shaking the sample to be identified to be uniform at room temperature to prepare a uniform dispersion liquid of the sample to be detected, adding 10-1000 mu L of the uniform dispersion liquid of the sample to a conventional Sabouraud's syndrome liquid culture medium containing 6-600mL of the uniform dispersion liquid of the sample, placing the mixture in a shaking culture at 200 +/-50 rpm at 15-35 ℃, and sampling the mixture after culturing for 6-24h to form a liquid to be detected of the sample to be identified.
According to the scheme, the samples are products such as farmlands, agricultural products, traditional Chinese medicinal materials, feeds and the like.
In the scheme, the aflatoxin early warning molecule nano antibody can be prepared by taking AFT-YJFZP008 as an immune antigen, immunizing alpaca in a conventional manner and utilizing a known conventional nano antibody preparation technical scheme.
In the scheme, the aflatoxin early warning molecule monoclonal antibody can be prepared by taking AFT-YJFZP008 as an immune antigen, immunizing a Balb/c mouse by a conventional mode and preparing a technical scheme by using a known conventional mouse-derived monoclonal antibody.
The aflatoxin early warning molecule rabbit polyclonal antibody can be prepared by taking AFT-YJFZP008 as an immune antigen, immunizing test rabbits such as New Zealand white rabbits in a conventional manner and the like, and then utilizing a known conventional rabbit polyclonal antibody preparation technical scheme.
The early aflatoxin pollution risk early warning intelligent sensing card can be used for identifying the aflatoxin production capacity of the aspergillus strains, and the higher the aflatoxin early warning molecule content in the strains to be identified is, the stronger the aflatoxin production capacity of the identification strains is, namely the production capacity is; the early aflatoxin pollution risk early warning intelligent sensing card can be used for identifying whether aflatoxin toxin-producing strains with strong toxin-producing capability exist in products such as farmlands, agricultural products, traditional Chinese medicinal materials, feeds and the like, and if the aflatoxin early warning molecule content in a sample to be identified is high, the sample to be identified contains the aflatoxin-producing strains with strong toxin-producing capability;
the strain virulence of the aspergillus flavus is an index for measuring the capability of the strain to produce the aflatoxin, and the stronger the strain virulence, the more the strain can produce the aflatoxin in the same time and under the same culture conditions. According to the early warning method, aflatoxin early warning molecules obtained by research are utilized, and a nano antibody or a monoclonal antibody of the aflatoxin toxigenic bacteria virulence early warning molecules and a rabbit-derived polyclonal antibody (the aflatoxin early warning molecules can be directly used as antigens to prepare the nano antibody or the monoclonal antibody and the rabbit-derived polyclonal antibody) are combined, and a sandwich immunodetection method is constructed by the antibodies, and finally the early warning intelligent perception card for aflatoxin pollution risk is assembled; in the application of the aflatoxin pollution early-stage risk early-stage warning intelligent perception card, (1) the aflatoxin early-stage warning molecule is used for determining the characteristic that the concentration is in positive correlation with the strain virulence generating capacity, so that the aflatoxin pollution capacity of the aspergillus strain and the application of identifying whether the aflatoxin virulence generating strain with strong virulence generating capacity exists in farmlands, agricultural products and feeds, and (2) the aflatoxin early-stage warning molecule is used for determining the characteristic that the concentration is in positive correlation with the aflatoxin pollution risk in the agricultural products, so that the aflatoxin pollution risk level in the agricultural products is intelligently sensed, the aflatoxin pollution early-stage warning intelligent perception card is practical and easy to popularize, key tongs and scientific basis are provided for timely discovering the aflatoxin pollution risk and early-prevention early-control, and the aflatoxin pollution early-stage early-prevention intelligent perception card has important significance for promoting high-quality development of agricultural industries and guaranteeing food safety.
The invention has the beneficial effects that:
1. the early aflatoxin pollution risk early warning intelligent perception card can be used for quickly and quantitatively detecting aflatoxin early warning molecules, AFT-YJFZP008 and 2 can be used for identifying and comparing the aflatoxin production capacity of aflatoxin production strains in aspergillus, 3 can be used for identifying whether aflatoxin production strains with strong production capacity exist in farmlands, agricultural products and feeds, 4 can be used for quickly and intelligently perceiving the aflatoxin pollution risk level in the agricultural products, 5 is easy to operate, strong in practicability and easy to popularize and apply, and 6 has important significance for promoting the high-quality development of agricultural industries and guaranteeing the food safety.
Detailed Description
Example 1 preparation of Aflatoxin early warning molecule AFT-YJFZP008
The culture medium is prepared according to the following formula: 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, pH6.5, and prepared to obtain a Chaudhur medium. Randomly selecting 10 strains of Aspergillus flavus distribution, virulence and infection research in typical peanut producing areas in China-Master thesis of Chinese academy of agricultural sciences, Zhang apricot, page 33, published toxigenic strains HLJ-1, HeNZY-2, HuBha-24, JXZS-29-2, LNct-6, GXfc-34, GDZJ-122-2, JSnnt-1, HuNdx-7, HBHA-8-17 and the like, respectively inoculating the strains into the above Hakken culture medium, culturing for 5 days at 28 ℃ and 200rpm/min, fully homogenizing by a conventional method, crushing cells, and purifying by a conventional protein purification system, protein electrophoresis, immunoaffinity and the like to obtain the aflatoxin early warning molecule AFT-YJFZP 008. Test results show that AFT-YJFZP008 can be prepared in all the strains of the toxigenic strains, under the same culture conditions, AFT-YJFZP008 prepared by HBHA-8-17 is the most abundant, and AFT-YJFZP008 prepared by HLJ-1 is the least abundant.
The immunoaffinity method is characterized in that an immunoaffinity column is prepared by using a nano antibody or a monoclonal antibody of aflatoxin early warning molecule AFT-YJFZP008 through a conventional method, and then the immunoaffinity column is enriched and purified from aflatoxin toxin-producing fungal cell crushing liquid by using an immunoaffinity method and is obtained by dissolving with deionized water. The immunoaffinity column is prepared by using aflatoxin early warning molecule AFT-YJFZP008 antibody through a conventional affinity column preparation scheme. Specifically, the aflatoxin toxigenic bacteria cell disruption solution can be diluted by using a sample solution, filtered by conventional filter paper, continuously added into the immunoaffinity column, washed by conventional eluent of the immunoaffinity column when the basic flow is exhausted, finally eluted by glycine buffer solution with pH 2.2 or 70% methanol aqueous solution, the solution is timely removed by a conventional ultrafiltration centrifugal method after the eluent is collected, and then the protein remained in an ultrafiltration centrifugal tube is dissolved out from the ultrafiltration centrifugal tube by using sterile water, so that aflatoxin early warning molecule AFT-YJFZP008 aqueous solution can be obtained.
An initial acquisition and excavation method of aflatoxin toxigenic bacteria virulence early warning molecule AFT-YJFZP008 comprises the following steps:
the method for discovering the aspergillus flavus strain to produce virulence early warning molecules comprises the following steps:
(1) culturing Aspergillus flavus strain with high virulence producing ability to obtain 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 an animal to be tested with the aspergillus flavus antigen to obtain a nano antibody library or a monoclonal antibody library;
(3) obtaining protein combined solutions of the aspergillus flavus strains with different virulence productions, detecting the proteins of the aspergillus flavus strains with different virulence productions 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 showing positive correlation with the virulence produced by the aspergillus flavus strain, namely an early warning molecular antibody for the virulence produced by the aspergillus flavus strain, and finding out a protein corresponding to the indication molecular antibody for the virulence produced by the aspergillus flavus strain, namely the early warning molecular antibody for the virulence produced by the developed aspergillus flavus strain.
In the scheme, the aspergillus flavus strain with strong virulence producing capacity in the step (1) is separated and identified from the nature by a conventional method or obtained by artificial modification, and the identification result of the strain with strong virulence producing capacity by an NY/T2311-2013 standard method is not less than 10 mug/kg.
The aspergillus flavus strains with different virulence generating capacities in the step (3) are not less than 3 strains, and the virulence generating capacity is identified by an NY/T2311-2013 standard method, and the results are presented in at least 3 levels of high, medium and low.
The culture medium adopted in the culture of the aspergillus flavus strain with strong virulence is a Chao's culture medium or other nutrients for 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 disruptor and the like.
The amount of the carbodiimide is 0.005-0.1 g per 1.0mL of combined extracellular secretion protein mixture and intracellular protein mixture.
The coupling reaction is carried out for 2-6 h at 15-37 ℃, and is carried out overnight at 4-10 ℃.
The immunization is a conventional immunization mode and is used for inoculating the aspergillus flavus antigen. The test animal is a white mouse or alpaca or other test animals with similar effects.
According to the scheme, the antibody preparation process refers to a conventional nano antibody preparation technical process or a conventional hybridoma monoclonal antibody preparation technical process based on cell fusion.
According to the scheme, the detection of the proteins of the aspergillus flavus strains with different virulence degrees is realized by adopting the conventional WesternBlot technical process, namely, the proteins of the aspergillus flavus strains with different virulence degrees 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 above scheme, the above direct method is that the antibody in the antibody library is coupled with a signal material by a conventional method, and then undergoes an immunological binding reaction with the corresponding protein transferred onto the nitrocellulose membrane.
According to the above scheme, the indirect method is that the antibody in the antibody library is firstly subjected to immunological binding reaction with the corresponding protein transferred to the nitrocellulose membrane, and then the second antibody and the signal material conjugate are subjected to immunological binding reaction with the antibody bound to the nitrocellulose membrane.
The signal material in the detection is horseradish peroxidase or colloidal gold or fluorescent material or other materials with similar effects. The detection signal is a chromogenic reaction signal or a spot signal or a fluorescent signal.
The aflatoxin early warning molecule AFT-YJFZP008 antibody can also be prepared by using all peptide fragments or partial peptide fragments of the aflatoxin early warning molecule AFT-YJFZP008 antibody through a conventional antibody preparation technical process after knowing the whole sequence of the aflatoxin early warning molecule AFT-YJFZP 008.
Example 2 Aflatoxin early warning molecule AFT-YJFZP008 nano antibody preparation
AFT-YJFZP008 is used as an immune antigen, an alpaca or Balb/c mouse is immunized by a conventional mode, and the preparation technical scheme of a known conventional nano antibody or mouse monoclonal antibody is utilized to prepare the alpaca or Balb/c mouse.
Dissolving AFT-YJFZP008 obtained by the preparation in a conventional PBS buffer solution or physiological saline until the concentration is not lower than 0.1mg/mL, mixing and emulsifying the solution with Freund complete adjuvant in the same volume, immunizing alpaca by a back subcutaneous or intradermal multipoint injection mode, then strengthening the immunity for 1 time every 2-4 weeks, and replacing the Freund complete adjuvant with Freund incomplete adjuvant when strengthening the immunity. Monitoring the immune effect by adopting a conventional ELISA process until the serum titer of the alpaca does not rise any more, then performing operations of venous blood sampling, total RNA extraction, cDNA synthesis, VHH gene amplification, VHH gene fragment recovery, VHH gene and pCANTAB 5E (his) vector double enzyme digestion treatment, connection product electrotransformation, construction of a nano antibody gene bank, rescue of the nano antibody gene bank and the like on the immune alpaca according to a method of patent document CN103866401A, and finally obtaining the rescued nano antibody gene bank.
Fixing AFT-YJFZP008 obtained by the preparation on a solid phase carrier such as a 96-hole enzyme label plate according to the gradient of 8 mu g/hole, 2 mu g/hole, 0.5 mu g/hole and 0.1 mu g/hole, carrying out elutriation on the rescued nano antibody gene bank for 2-4 times according to the method of patent document CN103866401A, identifying the antibody generated by each phage clone by AFT-YJFZP008 and indirect non-competitive ELISA, wherein the phage corresponding to the positive result is a phage positive clone, and preparing the nano antibody, namely the nano antibody of the AFT-YJFZP008, by the conventional method of preparing the nano antibody by using the positive clone, wherein the nano antibody is used for further application and research work, preferably, the nano antibody has strong specificity and high affinity by the representation of the ELISA method.
Example 3 Aflatoxin early warning molecule AFT-YJFZP008 monoclonal antibody preparation
AFT-YJFZP008 is used as an immune antigen, an alpaca or Balb/c mouse is immunized by a conventional mode, and the preparation technical scheme of a known conventional nano antibody or mouse monoclonal antibody is utilized to prepare the alpaca or Balb/c mouse.
Dissolving AFT-YJFZP008 obtained by the preparation in a conventional PBS buffer solution or physiological saline until the concentration is not lower than 0.1mg/mL, mixing and emulsifying the solution with Freund complete adjuvant in the same volume, performing back subcutaneous or intradermal multipoint injection on BALB/c mice, performing booster immunization for 1 time every 2-4 weeks, and replacing the Freund complete adjuvant with Freund incomplete adjuvant during booster immunization. Monitoring the immune effect by adopting a conventional ELISA process until the serum titer of a BALB/c mouse does not rise, then separating splenocytes of the immunized mouse, fusing the splenocytes with murine myeloma cells SP2/0, and selectively culturing hybridoma cells by using a semisolid culture medium by the method of reference patent document CN103849604A, and after white spots of a needle tip grow on the semisolid culture medium, respectively picking the white spots to a 96-well culture plate internally provided with a conventional hybridoma culture medium, thereby obtaining a monoclonal hybridoma resource library.
The monoclonal antibody is obtained from the culture supernatant of the monoclonal hybridoma by the method of reference patent document CN103849604A, the AFT-YJFZP008 obtained by the preparation is fixed on a solid phase carrier such as a 96-hole enzyme label plate according to gradients of 8 mug/hole, 2 mug/hole, 0.5 mug/hole and 0.1 mug/hole, each monoclonal antibody is identified by an indirect non-competitive ELISA program, and a positive clone is selected to obtain the AFT-YJFZP008 monoclonal antibody which is used for further application and research work, preferably the AFT-YJFZP008 monoclonal antibody with the characteristics of strong specificity and high affinity.
Example 4 Aflatoxin early warning molecule AFT-YJFZP008 rabbit source polyclonal antibody preparation
AFT-YJFZP008 is used as an immune antigen, a test rabbit such as a New Zealand white rabbit and the like is immunized by a conventional mode, and the test rabbit can be obtained by developing by utilizing a known conventional rabbit polyclonal antibody preparation technical scheme.
The AFT-YJFZP008 obtained by the preparation method is directly used as an antigen, a solution with the concentration of not less than 0.1mg/mL and Freund's complete adjuvant are mixed and emulsified in equal volume, the New Zealand white rabbits are boosted for 1 time every 2-4 weeks by a back subcutaneous or intradermal multipoint injection mode, and the Freund's complete adjuvant is replaced by the Freund's incomplete adjuvant during boosting. And (3) monitoring the immune effect by adopting a conventional ELISA process, and preparing the serum of the immune animal by a conventional method after the serum titer of the immune animal does not rise any more, namely the rabbit source polyclonal antibody of the aflatoxin early warning molecule AFT-YJFZP 008.
Example 5 Aflatoxin pollution early risk early warning intelligent perception card preparation
The aflatoxin pollution early-stage risk early-stage warning intelligent perception card is formed by assembling aflatoxin early-stage warning molecule nano antibody or aflatoxin early-stage risk early-stage warning molecule monoclonal antibody, aflatoxin early-stage risk early-stage warning molecule rabbit polyclonal antibody marked by a signal material, a sample pad, a nitrocellulose membrane, a water absorption pad, a detection pad and a sample pad are sequentially pasted on one surface of the bottom plate from top to bottom, adjacent pads are connected at joints in an overlapping manner, the nitrocellulose membrane is used as a base pad, a transverse quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with the goat anti rabbit antibody, the detection line is coated with the aflatoxin early-stage warning molecule nano antibody or the aflatoxin early-stage warning molecule monoclonal antibody, and the aflatoxin early-warning molecule polyclonal antibody marked by the signal material is loaded on the sample pad, or loaded separately, where the difference is the loading of the core reagents:
(1) the detection line of the nitrocellulose membrane is fixed with an aflatoxin early warning molecule nano antibody or an aflatoxin early warning molecule monoclonal antibody, and the fixing method comprises the following steps: spraying by using a conventional membrane spotting instrument, wherein the concentration of the aflatoxin early warning molecule nano antibody or the aflatoxin early warning molecule monoclonal antibody is 1mg/mL, and the spraying speed is 0.6 muL/cm; the quality control line is fixed with goat anti-rabbit antibody, and the fixing method comprises the following steps: spraying with a conventional membrane spotting instrument at a speed of 0.6mL/cm and a concentration of goat anti-rabbit antibody of 0.5 mg/mL.
(2) The aflatoxin early warning molecule rabbit polyclonal antibody marked by the signal material is loaded on a sample pad, or is independently loaded in vessels such as conventional small holes, small bottles and the like, and is prepared by conventional freeze drying treatment.
The preparation method of the aflatoxin early warning molecule rabbit polyclonal antibody marked by the signal material is illustrated by taking a conventional europium latex microsphere as an example as follows: taking 100 mu L europium latex microspheres, mixing uniformly in 900 mu L MES solution with pH of 6.0 by vortex, placing in an ultrasonic cell disruption instrument, performing centrifugation at the power of 20 percent and the ultrasonic frequency of 9s and 11000rpm for 10min, carefully discarding the supernatant, adding 1mL of boric acid buffer solution (pH 8.2) into the precipitate for resuspending, performing repeated ultrasonic frequency of 9s after uniform vortex mixing, adding 20 mu L of carbodiimide aqueous solution which is now prepared and is 15mg/mL for activation, violently shaking at room temperature for 15min, centrifuging at the 11000rpm for 10min, carefully discarding the supernatant, adding 1mL of boric acid buffer solution into the precipitate for resuspending, performing repeated ultrasonic frequency of 9s after uniform vortex mixing, adding 60 mu g of aflatoxin early warning molecule rabbit polyclonal antibody, shaking a table at the temperature of 10 ℃ for 2h, centrifuging at the 11000rpm for 10min, re-dissolving the precipitate with 1mL of 0.1 percent bovine serum albumin-phosphate buffer solution, repeating the ultrasonic step, and shaking a table at the temperature of 4 ℃ for 1h to seal the unbound sites on the surfaces of the microspheres by the bovine serum albumin; after the reaction is finished, the aflatoxin early warning molecule rabbit polyclonal antibody marked by the signal material can be obtained and stored at 4 ℃ for later use. When in use, the aflatoxin early warning molecule rabbit polyclonal antibody marked by the signal material obtained by the preparation is diluted 1000 times by adopting a conventional phosphate buffer solution containing 1% of sucrose, 0.5% of ovalbumin, 2.5% of tween-20 and 0.5% of PVPK30 for reuse.
(3) In mutual correspondence with the scheme (2) above, the following are: and a goat anti-rabbit antibody is fixed on the nitrocellulose membrane quality control line.
The aflatoxin early warning molecule AFT-YJFZP008 nano antibody or monoclonal antibody can be prepared by the methods of example 2 and example 3.
The aflatoxin early warning molecule AFT-YJFZP008 rabbit source polyclonal antibody can be prepared by the method of example 4.
The quality guarantee period research result of the aflatoxin pollution early risk early warning intelligent sensing card shows that the aflatoxin pollution early risk early warning intelligent sensing card can be stored for more than 6 months at 4 ℃.
Example 5 establishment of Standard Curve for Aflatoxin early Risk early Warning Smart cards to rapidly determine Aflatoxin early warning molecule AFT-YJFZP008
Taking the aflatoxin early risk early warning intelligent sensing card finally prepared from the europium latex microspheres as an example, the establishment process of the standard curve for rapidly determining the aflatoxin early warning molecule AFT-YJFZP008 by using the aflatoxin early risk early warning intelligent sensing card is explained.
The aflatoxin early warning molecule AFT-YJFZP008 prepared in the embodiment 1 is prepared into solutions with series of concentrations of 0.001, 0.01, 0.1, 1, 10, 100ng/mL and the like by using a conventional phosphate buffer solution, then the aflatoxin early warning molecule rabbit polyclonal antibody marked by a signal material for dissolving the aflatoxin pollution early risk early warning intelligent sensing card is sequentially used, the conventional immunochromatography reaction is carried out for 15min, finally, a fluorescence signal on the aflatoxin pollution early risk early warning intelligent sensing card is read, the fluorescence signal is used as a vertical coordinate, the AFT-YJFZP008 is used as a horizontal coordinate to draw a standard curve, and the result shows that the aflatoxin pollution early risk early warning intelligent sensing card can rapidly and quantitatively determine the content of the aflatoxin early warning molecule AFT-YJFZP008, the detection limit can reach 0.1ng/mL, and the correlation coefficient can reach 0.99. And then, evaluating the repeatability and the accuracy of the aflatoxin early warning molecule AFT-YJFZP008 by adopting a conventional method to evaluate the early risk warning intelligent perception card of aflatoxin pollution, wherein the repeatability evaluation result shows that: the coefficient of variation of the measurement result is below 10%, which shows that the repeatability of the measurement result of the method is good; the accuracy evaluation result shows that the aflatoxin early warning molecule AFT-YJFZP008 prepared in the example 1 is added into products such as peanuts, corns and rice, the recovery rate range is 86-103%, and the method shows that the measurement result has good accuracy. Therefore, the aflatoxin pollution early risk early warning intelligent perception card method for rapidly and quantitatively determining AFT-YJFZP008 has good detection sensitivity and accuracy.
Evaluation of method specificity: in order to evaluate the specificity of the immunodetection method of the aflatoxin early warning molecule AFT-YJFZP008, a plurality of strains of fungi such as fusarium oxysporum, aspergillus niger, aspergillus ochraceus, fusarium moniliforme and the like which have certain homology with the aspergillus flavus are selected for research, and the cell disruption solution of the fungus culture is detected, so that the determination result shows that the method has no obvious cross reaction on the protein of the fungi which have homology with the aspergillus flavus, and the established aflatoxin early warning molecule AFT-YJFZP008 immunodetection method has good specificity.
Example 7 Aflatoxin contamination early risk early warning intelligent perception card for fast perception and identification of comparative aflatoxin strains
Step one, preparing a solution to be detected of a strain to be identified: according to the strong and weak strain virulence, selecting 10 strains of toxin-producing strains HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, HeNZY-2, HuBha-24, JSont-1, HuNdx-7, GDZJ-108-19, HBHA-8-17 and the like which are published in the publication of research on distribution, virulence producing capability and infection of aspergillus flavus in typical peanut producing areas in China-the Master academic thesis of Chinese academy of agriculture, page 33, of authors of ZXingxing, and the like, culturing the strains to be identified in a Czochralski culture medium in the kit, wherein the culture environment temperature is 15-35 ℃, and the culture time is not less than 12h, and (3) fully homogenizing a mixture of the culture medium and the culture, and diluting by 1-10 times by using the sample diluent of the aflatoxin pollution early risk early warning intelligent sensing card to obtain a to-be-detected solution of the strain to be identified.
Secondly, measuring the liquid to be measured of the strain to be identified: and (3) determining the liquid to be detected by using an aflatoxin pollution early risk early warning intelligent sensing card determination scheme, and reading and calculating the concentration of AFT-YJFZP008 in a sample to be detected.
According to the results of the above documents, the virulence producing capability of 10 strains such as HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, HeNZY-2, HuBha-24, Jcnt-1, HuNdx-7, GDZJ-108-19, HBHA-8-17 and the like are as follows: 0 mug/L, 3.8 mug/L, 4.9 mug/L, 67.2 mug/L, 81.7 mug/L, 192.0 mug/L, 204.4 mug/L, 297.4 mug/L and 1027.5 mug/L, which shows that HBZHX-21 and HBXY-36 are non-toxigenic strains, HBHA-1-4 and GDZJ-6 are weak-toxigenic strains, HBHA-8-17 and GDZJ-108-19 are strong-toxigenic strains.
Thirdly, judging the identification result: the higher the aflatoxin early warning molecule AFT-YJFZP008 content in the strain to be identified, the stronger the aflatoxin production capability, i.e. the production toxicity, of the identification strain is. According to the aflatoxin pollution early risk early warning intelligent perception card and the application technical scheme thereof, the results of measuring the AFT-YJFZP008 concentration of 10 strains of HBZHX-21, HBXY-36, HBHA-1-4, GDZJ-6, HeNZY-2, HuBha-24, JSnnt-1, HuNdx-7, GDZJ-108-19, HBHA-8-17 and the like are as follows in sequence: 0ng/mL, 7.2ng/mL, 9.9ng/mL, 42.5ng/mL, 67.7ng/mL, 112.0ng/mL, 127.6ng/mL, 176.0ng/mL, 537.1ng/mL, with the same results indicating: HBZHX-21 and HBXY-36 are non-toxigenic strains, HBHA-1-4 and GDZJ-6 are weak toxigenic strains, and HBHA-8-17 and GDZJ-108-19 are strong toxigenic strains. The determination result is consistent with the strain virulence generating sequence published in the published literature, i.e., the distribution, virulence generating capacity and infection research of aspergillus flavus in typical peanut producing areas in China, and the identification results of strains with strong virulence generating capacity and strains with weak virulence generating capacity are consistent with the literature.
Example 8 fast perception and identification of a virulent strain producing aflatoxin in a field by using an early aflatoxin contamination risk early warning smart perception card
Step one, preparing a to-be-detected liquid of a to-be-identified sample: selecting 4 parts of peanut flowering phase rhizosphere soil samples such as Jilin, Liaoning, Jiangxi, Fujian and the like, and sequentially naming the samples as a soil sample-1, a soil sample-2, a soil sample-3 and a soil sample-4. Sequentially weighing farmland soil samples to be detected, uniformly crushing, transferring into the sample diluent of the kit, wherein the concentration is 0.5g/mL, and vibrating at room temperature to be uniform to prepare the sample diluent to be detected. And adding 50 mu L of the sample diluent to be detected into 30mL of the Chao's medium of the kit, placing the mixture at 28 ℃ and carrying out shake culture at 200rpm, and sampling after culturing for 24h to form a sample to be detected of a sample to be identified.
Step two, the determination of the sample to be identified and the solution to be determined: and (3) determining the liquid to be detected by using an aflatoxin pollution early risk early warning intelligent sensing card determination scheme, and reading and calculating the concentration of AFT-YJFZP008 in a sample to be detected.
Thirdly, judging the identification result: if the aflatoxin early warning molecule AFT-YJFZP008 in the sample to be identified has high concentration, the sample to be identified contains the strong-virulence-producing strain of aflatoxin. According to the kit and the application technical scheme thereof, the results of measuring the concentration of AFT-YJFZP008 in the soil samples-1, -2, -3 and-4 are as follows in sequence: 0.1ng/mL, 0.2ng/mL, 12.7ng/mL, 19.5 ng/mL. The result shows that taking 50 microliter of sample uniform dispersion liquid added into a culture medium containing 30mL as an example, the amount of AFT-YJFZP008 contained in each milliliter of culture liquid in the soil sample-1 and the soil sample-2 is below 1.0ng, the strain does not contain aflatoxin and has strong virulence, and the pollution risk after the production of the corresponding farmland flowers is very low; the amount of AFT-YJFZP008 contained in each milliliter of culture solution in the soil sample-3 and the soil sample-4 is more than 10ng/mL, the aflatoxin-containing strain with strong virulence is contained, and the pollution risk after the flower production in corresponding farmlands is higher.
Example 9 fast perception and identification of a highly virulent strain containing aflatoxin in an agricultural product by using an early aflatoxin contamination risk early warning smart perception card
Step one, preparing a to-be-detected liquid of a to-be-identified sample: selecting 4 parts of agricultural product samples such as peanut, corn, rice, wheat and the like, sequentially weighing the farmland soil sample to be detected, crushing the farmland soil sample, transferring the crushed farmland soil sample to the sample diluent of the kit, wherein the concentration of the sample diluent is 0.5g/mL, and vibrating the diluted farmland soil sample to be uniform at room temperature to prepare the sample diluent to be detected. And adding 100 mu L of the sample diluent to be detected into 50mL of the Chao's medium containing the kit, placing the mixture at 28 ℃ and carrying out shake culture at 200rpm, and sampling after culturing for 6h to form a sample to be detected of a sample to be identified.
Step two, the determination of the sample to be identified and the solution to be determined: and (3) determining the liquid to be detected by using an aflatoxin pollution early risk early warning intelligent sensing card determination scheme, and reading and calculating the concentration of AFT-YJFZP008 in a sample to be detected.
Thirdly, judging the identification result: if the aflatoxin early warning molecule AFT-YJFZP008 in the sample to be identified has high concentration, the sample to be identified contains the strong-virulence-producing strain of aflatoxin. According to the kit and the application technical scheme thereof, the measured AFT-YJFZP008 concentration results in peanut, corn, rice and wheat samples are as follows in sequence: 16.2ng/mL, 11.3ng/mL, 0.4ng/mL, 0 ng/mL. The result shows that the culture solution of peanut and corn contains more than 10ng of AFT-YJFZP008 per ml, and the peanut and corn contains aflatoxin-producing virulent strain, so that the pollution risk is high; the amount of AFT-YJFZP008 contained in each milliliter of culture solution in the rice sample is below 1.0, the rice sample does not contain a strong virulence-producing strain of aflatoxin, and the pollution risk is very low; the measured wheat sample contains 0 amount of AFT-YJFZP008 in each milliliter of culture solution, does not contain aflatoxin toxigenic strains, and basically has no aflatoxin pollution risk.
Example 10 fast perception and identification of a highly virulent strain containing aflatoxin in a feed by using an early aflatoxin contamination risk early warning smart perception card
Step one, preparing a to-be-detected liquid of a to-be-identified sample: selecting 4 parts of feed samples to be detected from the market, and sequentially naming the feed samples as feed-1, feed-2, feed-3 and feed-4. Sequentially weighing farmland soil samples to be detected, crushing the farmland soil samples, transferring the crushed farmland soil samples to the sample diluent of the kit, wherein the concentration of the sample diluent is 0.5g/mL, and vibrating the diluted farmland soil samples to be detected to be uniform at room temperature to prepare the sample diluent to be detected. And adding 50 mu L of the sample diluent to be detected into 30mL of the Chao's medium containing the kit, placing the mixture at 28 ℃ and carrying out shake culture at 200rpm, and taking a sample after culturing for 24h to form a sample to be detected of a sample to be identified.
Step two, the determination of the sample to be identified and the solution to be determined: and (3) determining the liquid to be detected by using an aflatoxin pollution early risk early warning intelligent sensing card determination scheme, and reading and calculating the concentration of AFT-YJFZP008 in a sample to be detected.
Thirdly, judging the identification result: if the aflatoxin early warning molecule AFT-YJFZP008 in the sample to be identified has high concentration, the sample to be identified contains the strong-virulence-producing strain of aflatoxin. According to the reagent kit and the application technical scheme thereof, the results of measuring the concentration of AFT-YJFZP008 in feed-1, feed-2, feed-3 and feed-4 are as follows in sequence: 0.5ng/mL, 19.6ng/mL, 10.7ng/mL, 72.2 ng/mL. The result shows that the amount of AFT-YJFZP008 contained in each milliliter of culture solution in the feed-1 sample is below 1.0ng/mL, the aflatoxin-free strong virulence-producing strain is not contained, and the pollution risk is very low; the feed-2, the feed-3 and the feed-4 contain more than 10ng/mL of AFT-YJFZP008 per milliliter of culture solution, and the strain contains aflatoxin with strong virulence, so that the pollution risk is higher after flower production in corresponding farmlands.
Example 11 correlation of aflatoxin-early warning molecule concentration in agricultural products with aflatoxin contamination level of agricultural products
293 parts of samples of agricultural products such as peanuts, corns, rice, wheat, walnuts, pistachios and the like are randomly selected, the concentration of aflatoxin early warning molecule AFT-YJFZP008 in the samples is measured by the method of example 5, and the aflatoxin level is measured by a high performance liquid chromatography-mass spectrometry combined method in GB 5009.22-2016 food safety national standard food determination of aflatoxin B group and G group. The measurement result shows that: when the measured concentration of AFT-YJFZP008 is less than 0.1 mu g/g, the aflatoxin pollution level of 90 percent of samples is below 1.0 mu g/kg, which is low risk; when the measured concentration of AFT-YJFZP008 is more than 1.0 mu g/g, the aflatoxin pollution level of a 75 percent sample is more than 10 mu g/kg, which is high risk; the other tests show that the concentration of AFT-YJFZP008 is 0.1-1.0 mug/g, which is a risk of stroke.
Further adopting a conventional Czochralski culture medium method to randomly culture 6 strains of aflatoxins from different production places to produce virulence strains, adopting the early warning molecule detection method and the aflatoxin detection method to track and monitor the elimination rule of AFT-YJFZP008 and aflatoxin in the culture process, finding that AFT-YJFZP008 can be detected after 16 hours of culture, and the aflatoxin is almost zero at the moment, wherein the result shows that the early warning molecule AFT-YJFZP008 is generated before the aflatoxin is generated.
Therefore, an aflatoxin molecule early warning method for agricultural products is established based on aflatoxin early warning molecules AFT-YJFZP008, when the measured concentration of AFT-YJFZP008 is less than 0.1 mu g/g, the risk is low, and in the aspect of guiding production, the measured agricultural products can be safely eaten or stored; when the measured concentration of AFT-YJFZP008 is more than 1.0 mu g/g, the risk is high, and in the aspect of guiding production, prevention and control and even reduction measures are required to prevent and control the pollution hazard of aflatoxin; when the concentration of the measured AFT-YJFZP008 is between 0.1 and 1.0 mu g/g, the measured agricultural products need to be continuously tracked and monitored for medium risk and production guidance. The method has the advantages that the accuracy of predicting the low risk of aflatoxin in agricultural products is up to 90%, and the accuracy of predicting the high risk is over 75%.
Embodiment 12 early warning of aflatoxin contamination risk level in agricultural products is quickly sensed and warned by using an intelligent sensing card for early warning of aflatoxin contamination risk
Agricultural products such as peanuts from 10 county-level peanut producing areas, corns from 3 county-level producing areas, rice from 3 county-level producing areas, wheat from 3 county-level producing areas, 1 walnut, 1 pistachio and the like are predicted by adopting the aflatoxin pollution early-stage risk early-warning intelligent sensing card. The prediction result shows that: 1) the concentration of AFT-YJFZP008 of peanuts in 1 county-level production area is more than 1.6 mu g/g, the risk is high, and effective aflatoxin prevention and control or reduction measures need to be taken for the product. 2) The concentration of AFT-YJFZP008 of 2 county-level producing area peanuts, 1 county-level producing area corn and 1 producing area walnut is in the range of 0.2-0.9 mu g/g, and is sequentially 0.53 mu g/mL, 0.29 mu g/mL, 0.85 mu g/mL and 0.26 mu g/mL, belonging to the medium risk and needing to continuously track and monitor the pollution risk.
In combination with the above specific examples, it is demonstrated that,
the technical scheme of the invention can be used for early warning the aflatoxin risk in agricultural products and guiding production in time, 1, the aflatoxin pollution early risk early warning intelligent sensing card for quickly detecting the aflatoxin early warning molecule immunity is provided, the aflatoxin pollution early risk early warning intelligent sensing card can be used for quickly and quantitatively detecting the aflatoxin early warning molecule AFT-YJFZP008 and 2, the aflatoxin pollution early warning intelligent sensing card can be used for identifying and comparing the aflatoxin production capacity of aflatoxin production strains in aspergillus, 3, whether the aflatoxin production strains with strong production toxicity exist in farmlands, agricultural products and feeds or not, 4, the aflatoxin pollution risk level in the agricultural products can be quickly and intelligently sensed, 5, the operation is easy, the practicability is strong, the popularization and the application are easy, and 6, the intelligent sensing card has important significance for promoting the high-quality development of agricultural industries and ensuring the food safety.
< 110 > institute of oil crops of Chinese academy of agricultural sciences
Less than 120, early aflatoxin pollution risk early warning intelligent sensing card 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 (8)

1. The aflatoxin pollution early-stage risk early-stage warning intelligent perception card is characterized by comprising an aflatoxin early-stage warning molecule nano antibody or an aflatoxin early-stage risk early-stage warning molecule monoclonal antibody, an aflatoxin early-stage warning molecule rabbit polyclonal antibody marked by a signal material, a sample pad, a nitrocellulose membrane, a water absorption pad, a bottom plate and a goat anti-rabbit antibody, wherein the water absorption pad, a detection pad and the sample pad are sequentially pasted on one surface of the bottom plate from top to bottom, adjacent pads are overlapped and connected at a joint, the detection pad takes the nitrocellulose membrane as a base pad, a transverse quality control line and a detection line are arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with the goat anti-rabbit antibody, the detection line is coated with the aflatoxin early-stage warning molecule nano antibody or the aflatoxin early-stage warning molecule monoclonal antibody, the aflatoxin early-stage risk early-stage warning molecule rabbit antibody marked by the signal material is loaded on the sample pad, or the aflatoxin early warning molecule is AFT-YJFZP008 peptide, and the amino acid sequence of the aflatoxin early warning molecule is shown in SEQ ID NO. 1.
2. The smart sensor card of claim 1, wherein: the signal material refers to conventional europium latex microspheres, gold nanoparticles and other marking materials capable of achieving similar effects.
3. The smart sensor card of claim 1, wherein: the aflatoxin early warning molecule rabbit polyclonal antibody marked by the signal material is a product obtained by coupling the signal material with the aflatoxin early warning molecule rabbit polyclonal antibody by a conventional marking method.
4. The application of the aflatoxin contamination early risk early warning intelligent sensing card of claim 1, which is characterized in that: the method is used for rapidly sensing the capability of producing aflatoxin by the aspergillus strain, identifying whether the aflatoxin producing strain with strong virulence exists in a sample or intelligently sensing the aflatoxin pollution risk level in agricultural products.
5. Use according to claim 4, characterized in that: the application method comprises the following steps: culturing and preparing a to-be-detected liquid of a strain to be identified or a sample to be identified by using a conventional Czochralski culture medium, and then determining the content of aflatoxin early warning molecules AFT-YJFZP008 by using the aflatoxin pollution early risk early warning intelligent perception card, wherein the specific method comprises the following steps: when the aflatoxin early warning molecule rabbit polyclonal antibody marked by the signal material is loaded on the sample pad, adding a strain to be identified or a liquid to be detected of a sample to be identified onto the sample pad of the aflatoxin pollution early risk early warning intelligent perception card, reacting for a period of time, and reading an analysis result;
the method comprises the steps of loading no aflatoxin early warning molecule rabbit polyclonal antibody marked by a signal material on a sample pad, adding a strain to be identified or a liquid to be detected of a sample to be identified into the aflatoxin early warning molecule rabbit polyclonal antibody marked by the signal material, then inserting an aflatoxin pollution early risk early warning intelligent sensing card into the strain to be identified or the liquid to be detected of the sample to be identified, reacting for a period of time, and reading an analysis result.
6. Use according to claim 4, characterized in that: the application method further comprises the following steps: and (3) evaluation of a detection result: according to the measurement result of the aflatoxin pollution early-stage risk early-stage warning intelligent perception card, obtaining the content of an aflatoxin warning molecule AFT-YJFZP008 in a liquid to be measured of a unit volume of a strain, analyzing the aflatoxin production capacity of the strain to be identified, wherein the higher the concentration of the aflatoxin warning molecule AFT-YJFZP008 in the strain to be identified is, the stronger the aflatoxin production capacity of the strain to be identified is, namely the production capacity is;
according to the measurement result of the aflatoxin pollution early-stage risk early-stage warning intelligent perception card, obtaining the content of aflatoxin warning molecule AFT-YJFZP008 in unit volume of sample liquid to be identified, and judging whether the sample to be identified contains a strong-virulence-producing strain of aflatoxin;
according to the measurement result of the aflatoxin pollution early-stage risk early-stage warning intelligent perception card, the content of aflatoxin warning molecules AFT-YJFZP008 in a unit volume of sample liquid to be identified is obtained, and the aflatoxin pollution risk in agricultural products is predicted.
7. Use according to claim 4, characterized in that: preparing a solution to be detected of the strain to be identified: culturing and diluting the strain to be identified to obtain a solution to be detected of the strain to be identified; the method specifically comprises the following steps: culturing the strain to be identified in a conventional Chaudou culture medium or other culture media suitable for the growth of the strain, wherein the culture environment temperature is 15-35 ℃, and the culture time is not less than 12 hours, then fully homogenizing the mixture of the culture medium and the culture, and diluting by 1-10 times with sterile water to obtain a solution to be detected of the strain to be identified;
preparing the sample solution to be identified: culturing a sample to be identified to obtain a liquid to be detected of the sample to be identified; the method specifically comprises the following steps: weighing a sample to be identified, transferring the sample to sterile water, shaking the sample to be identified to be uniform at room temperature to prepare a sample diluent to be detected, adding 10-1000 mu L of the sample diluent to a conventional Sabouraud's syndrome liquid culture medium containing 6-600mL of the sample diluent, placing the sample diluent in a conventional Sabouraud's syndrome liquid culture medium at 15-35 ℃ and shaking the sample at 200 +/-50 rpm for culture for 6-24h, and then sampling the sample to form the sample to be detected of the sample to be identified.
8. Use according to claim 4, characterized in that: the above samples are products of farmland, agricultural products, traditional Chinese medicinal materials, feed and the like.
CN202111041457.3A 2021-09-07 2021-09-07 Aflatoxin pollution early risk early warning intelligent perception card and application thereof Active CN113820491B (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2804866Y (en) * 2005-04-23 2006-08-09 江西中德大地生物工程有限公司 Non-toxin system immunity chromatography test paper for testing aflatoxin
WO2018161905A1 (en) * 2017-03-07 2018-09-13 中国农业科学院油料作物研究所 Time-resolved fluorescence immunochromatographic kit for simultaneous detection of mixed contamination of five mycotoxins such as aflatoxin b1 and application thereof
CN109557314A (en) * 2018-12-07 2019-04-02 中国农业科学院油料作物研究所 A method of virulence is produced for characterization and evaluation aflatoxicogenic strain
CN112305099A (en) * 2020-10-15 2021-02-02 中国农业科学院油料作物研究所 Early warning method before aflatoxin pollution
CN112530525A (en) * 2020-10-15 2021-03-19 中国农业科学院油料作物研究所 Aflatoxin pollution risk early warning molecule and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2804866Y (en) * 2005-04-23 2006-08-09 江西中德大地生物工程有限公司 Non-toxin system immunity chromatography test paper for testing aflatoxin
WO2018161905A1 (en) * 2017-03-07 2018-09-13 中国农业科学院油料作物研究所 Time-resolved fluorescence immunochromatographic kit for simultaneous detection of mixed contamination of five mycotoxins such as aflatoxin b1 and application thereof
CN109557314A (en) * 2018-12-07 2019-04-02 中国农业科学院油料作物研究所 A method of virulence is produced for characterization and evaluation aflatoxicogenic strain
CN112305099A (en) * 2020-10-15 2021-02-02 中国农业科学院油料作物研究所 Early warning method before aflatoxin pollution
CN112530525A (en) * 2020-10-15 2021-03-19 中国农业科学院油料作物研究所 Aflatoxin pollution risk early warning molecule and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
张杏 等: "中国西南花生产区黄曲霉菌分布、 产毒力及花生黄曲霉毒素污染", 《中国油料作物学报》, vol. 41, no. 5, pages 773 - 780 *
白艺珍 等: "油料作物主要生物毒素发生危害与检测控制研究", 《农产品质量与安全》, no. 6, pages 7 - 12 *

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