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Suspension chip detection method for simultaneously detecting four mycotoxins in corn and peanut

A mycotoxin and suspension chip technology, which is applied in measuring devices, biological testing, material inspection products, etc., can solve the problems of heavy workload, complicated operation, and long time consumption, and achieve the effect of small deviation

Inactive Publication Date: 2013-09-18
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the commonly used methods for detecting mycotoxins at home and abroad are mainly high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). The equipment is also more expensive; the latter is easy and fast to operate, but can only complete the detection of one mycotoxin at a time, and the detection of multiple toxins is not only time-consuming, laborious, and the workload is relatively large

Method used

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  • Suspension chip detection method for simultaneously detecting four mycotoxins in corn and peanut
  • Suspension chip detection method for simultaneously detecting four mycotoxins in corn and peanut
  • Suspension chip detection method for simultaneously detecting four mycotoxins in corn and peanut

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Coupling of four complete antigens of mycotoxins to microspheres with different codes

[0037] In the present invention, 22#, 26#, 30# and 32# carboxylated microspheres coded by Luminex Company are respectively coupled with four complete antigens of AFT-BSA, DON-BSA, T-2-BSA and ZEN-BSA.

[0038] 1.1 Activation of carboxylated microspheres

[0039] Place the brown vial containing the microspheres at room temperature for 30 min before coupling with the kit. During the experiment, aluminum foil was used to wrap the outside of the tube. The sample should be added quickly to ensure that the light in the room is not too bright, and try to prevent the microspheres from being exposed to light for a long time to reduce the chance of exposure. Before activation, take EDC and S-NHS out of the -20°C refrigerator, aliquot them in small quantities, take out one tube each time they are used, and put them in a desiccator at room temperature for 1 hour in advance. If the ex...

Embodiment 2

[0056] Example 2 Determination of optimal working concentrations of four mycotoxin monoclonal antibodies and biotinylated secondary antibodies, and verification of specificity between antigens and antibodies

[0057] Use the microspheres coupled with the complete antigen to determine the optimal working concentration of the four mycotoxin monoclonal antibodies and biotinylated secondary antibodies to be detected. The specific steps are as follows:

[0058] (1) First prepare four mycotoxin monoclonal antibodies: AFT-Ab (concentrations are 25, 50, 100 and 200 ng / mL in sequence), DON-Ab (concentrations are 2.5, 5, 10 and 20 ng / mL in sequence), T-Ab 2-Ab (concentrations are 6.25, 12.5, 25 and 50ng / mL) and ZEN-Ab (concentrations are 50, 100, 200 and 400ng / mL), that is, four concentrations each, and combined with biotinylated goat anti-small Mouse secondary antibody (Sec-Ab) concentration (sequentially 250, 500, 1000 and 2000ng / mL) solution was titrated by checkerboard, and it was m...

Embodiment 3

[0064] The establishment of multi-channel standard curve when embodiment 3 detects four kinds of mycotoxins simultaneously

[0065] The experiments at this stage were carried out using the microspheres coupled with the complete antigen, and the determined optimal working concentrations of the four mycotoxin monoclonal antibodies with high specificity and biotinylated secondary antibodies to be detected.

[0066] Specific steps are as follows:

[0067] (1) Prepare the best working concentration solutions of four kinds of mycotoxin monoclonal antibodies, namely AFT-Ab 100ng / mL, DON-Ab 10ng / mL, T-2-Ab 25ng / mL, ZEN-Ab 200ng / mL, and Mix them in a PCR tube to ensure a total volume of 50-70 μL after mixing. Then, 0.5-0.7 μL of each of the four complete antigen-microsphere conjugates after high-speed vortex oscillation was added in sequence (to ensure that the number of each type of microspheres in each reaction well is >2000), and a blank control, negative For the control and posi...

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Abstract

The invention discloses a suspension chip detection method for simultaneously detecting various mycotoxins (aflatoxin B1, vomitoxin, T-2 toxin, and zearalenone). Complete antigens of the mycotoxins and carboxylated microspheres with different codes are coupled through an EDC method, and covalent binding is allowed. With a competition method, a suspension chip technology research is carried out. During detection, the conjugate and monoclonal antibodies of the mycotoxins are oscillated; standard products are added, such that corresponding small molecules of a detection target and the complete antigens compete for the limited monoclonal antibodies; centrifugation is carried out, and supernatant is removed; a biotinylated secondary antibody is added; centrifugation is carried out, and supernatant is removed; SA-PE is added, such that a composition of detection target antigen-monoclonal antibody-secondary antibody-SA-PE is obtained; a median fluorescence value is reduced with the increasing of the concentration of the small molecules, such that a multichannel competition standard curve is drawn. Methanol / water with a certain ratio is used for pre-treating peanut and corn, and multichannel standard curves in corn and peanut are respectively drawn with a negative treatment liquid as a diluting liquid. Through the researches on matrix effect, method accuracy, and method repeatability, various mycotoxins in corn and peanut can be simultaneously detected with the suspension chip method. With the process, a sensitivity reaches a pg-ng level, the result is stable and reliable, sample dose is low, and the method is simple and fast. According to the invention, a novel method is provided for the detection of various mycotoxins.

Description

technical field [0001] The present invention provides a kind of suspension chip method that can detect four kinds of mycotoxins (comprising aflatoxin B1, deoxynivalenol, T-2 toxin and zearalenone) at the same time, particularly relate to a kind of simultaneous detection polysaccharides in corn and peanut Suspension chip detection technology for a mycotoxin. Background technique [0002] Fungi (Fungi) are a class of microorganisms that have cell walls, do not contain chlorophyll, have no roots, leaves and stems, live in a saprophytic or parasitic manner, and can reproduce sexually or asexually. Mycotoxins, also known as mycotoxins, are metabolites or secondary metabolites produced by certain fungi. There are currently more than 350 different mycotoxins known. Usually, mycotoxins contaminate food crops and animal feed. After entering the biological chain, they can not only lead to mildew and deterioration of agricultural products, loss of nutritional components, and lower pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577
Inventor 高志贤王莹宁保安吕志强孙智勇柳明彭媛范献军董建伟周彩虹
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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